Journal of Molecular Biology

The metalloregulatory zinc site in Streptococcus pneumoniae AdcR, a zinc-activated MarR-family repressor.

Publication Date: 2010 Aug 27 PMID: 20804771
Authors: Reyes-Caballero, H. - Guerra, A. J. - Jacobsen, F. E. - Kazmierczak, K. M. - Cowart, D. - Koppolu, U. M. - Scott, R. A. - Winkler, M. E. - Giedroc, D. P.
Journal: J Mol Biol

Streptococcus pneumoniae D39 AdcR (adhesin competence repressor) is the first metal-sensing member of the MarR (multiple antibiotic resistance repressor) family to be characterized. Expression profiling with a DeltaadcR strain grown in liquid culture (brain heart infusion; BHI) under microaerobic conditions reveals upregulation of 13 genes including adcR and adcCBA, encoding a high affinity ABC uptake system for zinc, and genes encoding cell-surface zinc-binding pneumococcal histidine triad (Pht) proteins and AdcAII (Lmb, laminin binding). The DeltaadcR, H108Q and H112Q adcR mutant allelic strains grown in 0.2 mM Zn(II) exhibit a slow-growth phenotype and a approximately 2-fold increase in cell-associated Zn(II). Apo-and Zn(II)-bound AdcR are homodimers in solution and binding to a 28-mer DNA containing an adc operator is strongly stimulated by Zn(II) with K(DNA-Zn)= 2.4 x10(8) M(-1) (pH 6.0, 0.2 M NaCl, 25 masculineC). AdcR binds two Zn(II) per dimer, with step-wise Zn(II) affinities K(Zn1) and K(Zn2) of >/=10(9) M(-1) at pH 6.0 and >/=10(12) M(-1) at pH 8.0. X-ray absorption spectroscopy (XAS) of the high affinity site reveals a pentacoordinate N/O complex and no cysteine coordination, the latter finding corroborated by wild-type-like functional properties of C30A AdcR. Alanine substitution of conserved residues His42 in the DNA binding domain, and His108 and His112 in the C-terminal regulatory domain, abolish high affinity Zn(II) binding and greatly reduce Zn(II)-activated binding to DNA. NMR studies reveal that these mutants adopt the same folded conformation as dimeric wild-type apo AdcR, but fail to conformationally switch upon Zn(II) binding. These studies clearly identify His42, His108 and H112 as metalloregulatory zinc ligands in S. pneumoniae AdcR.

post to: CiteULike

Structural characterization of protein-protein complexes by integrating computational docking with small-angle scattering data.

Publication Date: 2010 Aug 27 PMID: 20804770
Authors: Pons, C. - D'Abramo, M. - Svergun, D. I. - Orozco, M. - Bernado, P. - Fernandez-Recio, J.
Journal: J Mol Biol

X-ray crystallography and NMR can provide detailed structural information of protein-protein complexes, but technical problems make their application in the high-throughput regime still challenging. Other methods such as Small-Angle X-ray Scattering (SAXS) are more promising for large-scale application, but at the cost of lower resolution, a problem that can be solved by complementing SAXS data with theoretical simulations. In this study we propose a novel strategy that combines SAXS data and accurate protein-protein docking simulations. The approach has been benchmarked on a large pool of known structures with synthetic SAXS data, and on three experimental examples. The combined approach (pyDockSAXS) provided a significantly better success rate (43% for the top 10 predictions) than the two methods when applied separately. Further analyses of the influence of different docking parameters made it possible to further increase success rates for specific cases, and to define guidelines for improving the data-driven protein-protein docking protocols.

post to: CiteULike

Effects of Turn Stability and Side Chain Hydrophobicity on the Folding of b-structures.

Publication Date: 2010 Aug 27 PMID: 20804769
Authors: Shao, Q. - Wei, H. - Gao, Y. Q.
Journal: J Mol Biol

Key elements of the b-structure folding include the hydrophobic core collapse, the turn formation and the assembling of backbone hydrogen bonds. In the present folding simulations of several b-hairpins and b-sheets (peptide 1, GB1, TRPZIP2, TRPZIP4, 20mer and 20mer(D)P6D), the folding free energy landscape as the function of several reaction coordinates corresponding to the three key elements indicates apparent dependence on the turn stability and the side chain hydrophobicity, which demonstrates different folding mechanisms of similar b-structures of varied sequence. It is found that the turn stability is the key factor to determine the formation order of the three structural elements in the folding of b-structure. Moreover, the turn stability and the side chain hydrophobicity both affect the stability of backbone hydrogen bonds. The three-stranded b-sheets fold through a three-state transition in which the formation of one hairpin always takes precedence over the other. The different stability of two anti-parallel hairpins in each three-stranded b-sheet is shown to correlate well with the different level of their hydrophobic interactions.

post to: CiteULike

Conformational exchange is critical for the productivity of an oxidative folding intermediate with buried free cysteines.

Publication Date: 2010 Aug 27 PMID: 20804768
Authors: Gross, G. - Gallopin, M. - Vandame, M. - Couprie, J. - Stura, E. - Zinn-Justin, S. - Drevet, P.
Journal: J Mol Biol

Much has been learned about the folding of proteins from comparative studies of the folding of proteins that are related in sequence and structure. Observation of the effects of mutations helps account for sequence-specific properties and large variations in folding rates observed in homologous proteins, which are not explained by structure-derived descriptions. The folding kinetics of variants of a b-stranded protein, toxin a from Naja nigricollis, depends on the length of their loop lk1. These proteins, named Tox60, Tox61 and Tox62, contain four disulfide bonds. We show that their oxidative refolding pathways are similar. Differences in these pathways are restricted to the last step of the reaction i.e. the closure of the last disulfide. At this step, two species of three disulfide intermediates are observed: intermediate C lacking the B3 disulfide and intermediate D lacking the B2 disulfide. Surprisingly, D is the most productive intermediate for Tox61 despite the low accessibility of its free cysteines. However, in the case of Tox62, its conversion efficiency drops by two orders of magnitude and C becomes the most productive intermediate. NMR was used in order to study the structural dynamics of each of these intermediates. Both three disulfide intermediates of Tox61 exist in two forms exchanging on the 1-100 millisecond scale. One of these forms is structurally very close to the native Tox61 whereas the other is always significantly more flexible on a picosecond to nanosecond timescale. On the other hand, in the case of Tox62, the three disulfide intermediates only show a native-like structure. The higher conformational heterogeneity of Tox61 intermediate D allows an increased accessibility of its free cysteines to oxidative agents, which explains its faster native disulfide formation. Thus, residue deletion in loop lk1 probably abrogates stabilizing intramolecular interactions, creates conformational heterogeneity and increases the folding rate of Tox60 and Tox61 compared to Tox62.

post to: CiteULike

Structure of a Longitudinal Actin Dimer Assembled by Tandem W Domains - Implications for Actin Filament Nucleation.

Publication Date: 2010 Aug 27 PMID: 20804767
Authors: Rebowski, G. - Namgoong, S. - Boczkowska, M. - Leavis, P. C. - Navaza, J. - Dominguez, R.
Journal: J Mol Biol

Actin filament nucleators initiate polymerization in cells in a regulated manner. A common architecture among these molecules consists of tandem W domains that recruit three to four actin subunits to form a polymerization nucleus. We describe a low-resolution crystal structure of an actin dimer assembled by tandem W domains, where the first W domain is crosslinked to Cys-374 of the actin subunit bound to it, whereas the last W domain is followed by the C-terminal pointed end-capping helix of Tbeta4. While the arrangement of actin subunits in the dimer resembles that of a long-pitch helix of the actin filament, important differences are observed. These differences result from steric hindrance of the W domain with inter-subunit contacts in the actin filament. We also determined the structure of the first W domain of Vibrio parahaemolyticus VopL crosslinked to actin Cys-374, and show it to be nearly identical to non-crosslinked W-actin structures. This result validates the use of crosslinking as a tool for the study of actin nucleation complexes, whose natural tendency to polymerize interferes with most structural methods. Combined with a biochemical analysis of nucleation, the structures may explain why nucleators based on tandem W domains with short inter-W linkers have relatively weak activity, cannot stay bound to filaments after nucleation, and are unlikely to influence filament elongation. The findings may also explain why Nucleation Promoting Factors of the Arp2/3 complex, which are related to tandem W domain nucleators, are ejected from branch junctions after nucleation. We finally show that the simple addition of the C-terminal pointed end-capping helix of Tbeta4 to tandem W domains can change their activity from actin filament nucleation to monomer sequestration.

post to: CiteULike

Probing the Transition State of the Allosteric Pathway of the Shaker Kv Channel Pore by Linear Free Energy Relations.

Publication Date: 2010 Aug 27 PMID: 20804766
Authors: Azaria, R. - Irit, O. - Ben-Abu, Y. - Yifrach, O.
Journal: J Mol Biol

Long-range coupling between distant functional elements of proteins may rely on allosteric communication trajectories lying along the protein structure, as described in the case of the Shaker voltage-activated potassium channel (Kv) model allosteric system. Communication between the distant Kv channel activation and slow inactivation pore gates was shown to be mediated by a network of local pairwise and higher-order interactions among the functionally-unique residues comprising the allosteric trajectory. The mechanism by which conformational changes propagate along the Kv channel allosteric trajectory to achieve pore opening remains, however, unclear. Such conformational changes may propagate in either a concerted or a sequential manner during the reaction coordinate of channel opening. To discriminate between these possibilities, residue-level structural information on the transition state of channel gating is required. Here, we combine patch clamp electrophysiology recordings of Kv channel gating and analysis using linear free energy relations, focusing on a select set of residues spanning the allosteric trajectory of the Kv channel pore. We show that all allosteric trajectory residues tested exhibit an open-like conformation in the transition state of channel opening, implying that coupling interactions occur along the trajectory break in a concerted manner upon moving from the closed to the open state. Energetic coupling between the Kv channel gates thus occurs in a concerted fashion in both the spatial and temporal dimensions, strengthening the notion that such trajectories correspond to pathways of mechanical deformation along which conformational changes propagate.

post to: CiteULike

Crystal Structures of Nucleosome Core Particles Containing the '601' Strong Positioning Sequence.

Publication Date: 2010 Aug 25 PMID: 20800598
Authors: Vasudevan, D. - Chua, E. Y. - Davey, C. A.
Journal: J Mol Biol

By defining histone-DNA context and modulating access to specific sites, nucleosome positioning plays a key role in genomic regulation. Moreover, histone-DNA register influences double helical structure, which in turn can affect small molecule and protein factor association. Analysis of genomic and synthetic DNA has revealed sequence motifs that direct nucleosome positioning in vitro, and thus establishing the basis for the DNA sequence dependence of positioning would shed light on the mechanics of the double helix and its contribution to chromatin structure in vivo. However, acquisition of well diffracting nucleosome core particle (NCP) crystals is extremely dependent on the DNA fragment used for assembly, and all previous NCP crystal structures have been based on human alpha-satellite sequences. Here, we describe the crystal structures of Xenopus NCPs containing one of the strongest known histone octamer-binding and positioning sequences- the so-called '601' DNA. Two distinct 145 bp 601 crystal forms display the same histone-DNA register, which coincides with the occurrence of DNA stretching-overtwisting in both halves of the particle around five double helical turns from the nucleosome center, giving the DNA an 'effective length' of 147 bp. As we have found previously with stretching around two turns from the nucleosome center for a centromere-based sequence, the terminal stretching observed in the 601 constructs is associated with extreme kinking into the minor groove at purine-purine (pyrimidine-pyrimidine) dinucleotide steps. In other contexts, these step types display overall nonflexible behaviour, which raises the possibility that DNA stretching in the nucleosome or extreme distortions in general have unique sequence dependency characteristics. Our findings indicate that DNA stretching is an intrinsically predisposed, site-specific property of the nucleosome and suggest how to obtain NCP crystal structures with diverse DNA sequences.

post to: CiteULike

Crystallographic snapshot of glycosylasparaginase precursor poised for autoprocessing.

Publication Date: 2010 Aug 25 PMID: 20800597
Authors: Wang, Y. - Guo, H. C.
Journal: J Mol Biol

Glycosylasparaginase (GA) belongs to a family of N-terminal nucleophile (Ntn) hydrolases that autoproteolytically generate their mature enzymes from single chain protein precursors. Previously, based on a precursor structure paused at pre-autoproteolysis stage by a reversible inhibitor (glycine), we proposed a mechanism of intramolecular autoproteolysis. A key structural feature has been identified in which a highly strained conformation at the scissile peptide bond was hypothesized to be critical to drive the autoproteolysis through an N-O acyl shift. To examine this "twist-and-break" hypothesis, we report here a 1.9 A-resolution structure of an autoproteolysis-active precursor (a T152C mutant) that is free of inhibitor or ligand and is poised to undergo the autoproteolysis. The current crystallographic study has provided direct evidence for the natural conformation of the GA autocatalytic site without influence from any inhibitor or ligand. This finding has confirmed our previous proposal that the conformational strain is an intrinsic feature of an active precursor.

post to: CiteULike

Expression system-dependent modulation of HIV-1 envelope glycoprotein antigenicity and immunogenicity.

Publication Date: 2010 Aug 24 PMID: 20800070
Authors: Kong, L. - Sheppard, N. C. - Stewart-Jones, G. B. - Robson, C. L. - Chen, H. - Xu, X. - Krashias, G. - Bonomelli, C. - Scanlan, C. N. - Kwong, P. D. - Jeffs, S. A. - Jones, I. M. - Sattentau, Q. J.
Journal: J Mol Biol

Recombinant expression systems differ in the type of glycosylation they impart on expressed antigens such as the Human Immunodeficiency Virus Type-1 (HIV-1) envelope glycoproteins, potentially affecting their biological properties. We performed head-to-head antigenic, immunogenic and molecular profiling of two distantly-related Env surface (gp120) antigens produced in different systems: a) mammalian (293F) cells in the presence of kifunensine which impart only high mannose glycans; b) insect (Spodoptera frugiperda, Sf9) cells, which confer mainly paucimannosidic glycans; c) Sf9 cells recombinant for mammalian glycosylation enzymes (Sf9 Mimic), which impart high mannose, hybrid and complex glycans without sialic acid; d) 293F cells, which impart high mannose, hybrid and complex glycans with sialic acid. Molecular models revealed a significant difference in gp120 glycan coverage between the Sf9- and wild-type mammalian cell-derived material that is predicted to impact upon ligand binding sites proximal to glycans. Modelling of solvent-exposed surface electrostatic potentials showed that sialic acid imparts a significant negative surface charge that may influence gp120 antigenicity and immunogenicity. Gp120 expressed in systems that do not incorporate sialic acid displayed increased ligand binding to the CD4-binding and CD4-induced sites compared to those expressed in the system that do, and imparted other more subtle differences in antigenicity in a gp120 subtype-specific manner. Non-sialic acid-containing gp120 was significantly more immunogenic than the sialyated version when administered in two different adjuvants, and induced higher titres of antibodies competing for CD4 binding site ligand-gp120 interaction. These findings suggest that non-sialic acid imparting systems yield gp120 immunogens with modified antigenic and immunogenic properties, considerations which should be considered when selecting expression systems for glycosylated antigens to be used for structure/function studies and for vaccine use.

post to: CiteULike

Structural Analysis of HMGD-DNA Complexes Reveal Influence of Intercalation on Sequence Selectivity and DNA Bending.

Publication Date: 2010 Aug 24 PMID: 20800069
Authors: Churchill, M. E. - Klass, J. - Zoetewey, D. L.
Journal: J Mol Biol

The ubiquitous eukaryotic High-Mobility-Group-Box (HMGB) chromosomal proteins promote many chromatin-mediated cellular activities through their non-sequence-specific binding and bending of DNA. Minor groove DNA binding by the HMG box results in substantial DNA bending toward the major groove owing to electrostatic interactions, shape complementarity and DNA intercalation that occurs at two sites. Here, the structures of the complexes formed with DNA by a partially DNA intercalation-deficient mutant of Drosophila melanogaster HMGD have been determined by X-ray crystallography at a resolution of 2.85 A. The six proteins and fifty base pairs of DNA in the crystal structure revealed a variety of bound conformations. All of the proteins bound in the minor groove, bridging DNA molecules, presumably because these DNA regions are easily deformed. The loss of the primary site of DNA intercalation decreased overall DNA bending and shape complementarity. However, DNA bending at the secondary site of intercalation was retained and most protein-DNA contacts were preserved. The mode of binding resembles the HMGB1-boxA-cisplatin-DNA complex, which also lacks a primary intercalating residue. This study provides new insights into the binding mechanisms used by HMG boxes to recognize varied DNA structures and sequences as well as modulate DNA structure and DNA bending.

post to: CiteULike

The switch that does not flip - the blue-light receptor YtvA from Bacillus subtilis adopts an elongated dimer conformation independent of the activation state as revealed by a combined AUC and SAXS study.

Publication Date: 2010 Aug 24 PMID: 20800068
Authors: Jurk, M. - Dorn, M. - Kikhney, A. - Svergun, D. - Gartner, W. - Schmieder, P.
Journal: J Mol Biol

Photoreceptors play an important role in plants and bacteria by converting extracellular stimuli into intracellular signals. One distinct class are the blue-light sensitive phototropins harbouring a light, oxygen, voltage (LOV) domain coupled to various effector domains. Photon-absorption by the chromophore within the LOV domain results in an activation of the output domain via mechanisms that are hitherto not well understood. The photoreceptor YtvA from Bacillus subtilis is a bacterial analog of phototropins, consists of a LOV and a sulfate transporter/anti-sigma-factor antagonist (STAS) domain and is involved in the response of the bacterium to environmental stress. We present here analytical ultracentrifugation studies and small-angle x-ray scattering experiments, showing that YtvA is a dimer. On the basis of these results, we present a low-resolution model of the dimer in the dark and the lit state of the protein. In addition, we show that YtvA does neither change its oligomerization state nor its overall shape upon light activation.

post to: CiteULike

Conserved hydrophobic clusters on the surface of the Caf1A usher C-terminal domain are important for F1 antigen assembly.

Publication Date: 2010 Aug 23 PMID: 20797400
Authors: Dubnovitsky, A. P. - Duck, Z. - Kersley, J. E. - Hard, T. - Macintyre, S. - Knight, S. D.
Journal: J Mol Biol

The outer membrane usher protein Caf1A of the plague pathogen Yersinia pestis is responsible for the assembly of a major surface antigen, the F1 capsule. The F1 capsule is mainly formed by thin linear polymers of Caf1 protein subunits. The Caf1A usher promotes polymerization of the subunits and secretion of the growing polymers to the cell surface. The usher monomer (811 aa, 90.5 kDa) consists of a large transmembrane beta-barrel forming a secretion channel and three soluble domains. The periplasmic N-terminal domain binds chaperone:subunit complexes supplying new subunits for the growing fibre. The middle domain, which is structurally similar to Caf1 and other fimbrial subunits serves as a plug that regulates permeability of the usher. Here, we describe identification, characterization and crystal structure of a third soluble domain of the usher comprising the C-terminal 89 residues of Caf1A (Caf1A(C)). Caf1A(C) is shown to be a periplasmic domain with a seven-stranded beta-barrel fold. Analysis of C-terminal truncation mutants of Caf1A demonstrated that presence of the Caf1A(C) domain is crucial for function of the usher in vivo, but that it is not required for the initial binding of the chaperone:subunit complexes to the usher. Two clusters of conserved hydrophobic residues on the surface of Caf1A(C) were found to be essential for efficient assembly of surface polymer. These clusters are conserved between the FGL and FGS families of chaperone-usher systems.

post to: CiteULike

Diversity of Bisubstrate Binding Modes of Adenosine Analogue-Oligoarginine Conjugates in Protein Kinase A and Implications for Protein Substrate Interactions.

Publication Date: 2010 Aug 21 PMID: 20732331
Authors: Pflug, A. - Rogozina, J. - Lavogina, D. - Enkvist, E. - Uri, A. - Engh, R. A. - Bossemeyer, D.
Journal: J Mol Biol

Crystal structures of the catalytic subunit alpha of cAMP-dependent protein kinase (PKAc) with three adenosine analogue-oligoarginine conjugates (ARCs) are presented. The rationally designed ARCs include moieties that, in combination, target both the ATP- and the peptide-substrate-binding sites of PKAc, thereby taking advantage of high-affinity binding interactions offered by the ATP site while utilizing an additional mechanism for target specificity via binding to the peptide substrate site. The crystal structures demonstrate that, in accord with the previously reported bisubstrate character of ARCs, the inhibitors occupy both binding sites of PKAc. Further, they show new binding modes that may also apply to natural protein substrates of PKAc, which have not been revealed by previous crystallographic studies. The crystal structures described here contribute to the understanding of the substrate-binding patterns of PKAc and should also facilitate the design of inhibitors targeting PKAc and related protein kinases.

post to: CiteULike

Optimal Packaging of FIV Genomic RNA Depends upon a Conserved Long-range Interaction and a Palindromic Sequence within gag.

Publication Date: 2010 Aug 21 PMID: 20732330
Authors: Rizvi, T. A. - Kenyon, J. C. - Ali, J. - Aktar, S. J. - Phillip, P. S. - Ghazawi, A. - Mustafa, F. - Lever, A. M.
Journal: J Mol Biol

The feline immunodeficiency virus (FIV) is a lentivirus that is related to human immunodeficiency virus (HIV), causing a similar pathology in cats. It is a potential small animal model for AIDS and the FIV-based vectors are also being pursued for human gene therapy. Previous studies have mapped the FIV packaging signal (psi) to two or more discontinuous regions within the 5' 511 nt of the genomic RNA and structural analyses have determined its secondary structure. The 5' and 3' sequences within psi region interact through extensive long-range interactions (LRIs), including a conserved heptanucleotide interaction between R/U5 and gag. Other secondary structural elements identified include a conserved 150 nt stem-loop (SL2) and a small palindromic stem-loop within gag open reading frame that might act as a viral dimerization initiation site. We have performed extensive mutational analysis of these sequences and structures and ascertained their importance in FIV packaging using a trans-complementation assay. Disrupting the conserved heptanucleotide LRI to prevent base pairing between R/U5 and gag reduced packaging by 2.8-5.5 fold. Restoration of pairing using an alternative, non-wild type (wt) LRI sequence restored RNA packaging and propagation to wt levels, suggesting that it is the structure of the LRI, rather than its sequence, that is important for FIV packaging. Disrupting the palindrome within gag reduced packaging by 1.5-3-fold, but substitution with a different palindromic sequence did not restore packaging completely, suggesting that the sequence of this region as well as its palindromic nature is important. Mutation of individual regions of SL2 did not have a pronounced effect on FIV packaging, suggesting that either it is the structure of SL2 as a whole that is necessary for optimal packaging, or that there is redundancy within this structure. The mutational analysis presented here has further validated the previously predicted RNA secondary structure of FIV psi.

post to: CiteULike

Intertwined Structured and Unstructured Regions of exRAGE Identified by Monitoring Hydrogen-Deuterium Exchange.

Publication Date: 2010 Aug 21 PMID: 20732329
Authors: Kupniewska-Kozak, A. - Gospodarska, E. - Dadlez, M.
Journal: J Mol Biol

Receptor for advanced glycation end products (RAGE) is a multiligand receptor that is engaged in many pathological processes. Potentially beneficial modification of its activity requires sound knowledge of its structural properties. However, up to now, only the structures of its separated domains have been published or deposited in databases. In this work, we used hydrogen-deuterium exchange and mass spectrometry to gain insight into the structural properties of exRAGE (extracellular region of RAGE)-the full extracellular part of the protein. The present work indicates the common and disparate features of full exRAGE as compared to the structural models of its separate domains. The highlight of the present study is the contrasting behavior of the different regions of the protein, with the protected regions neighboring fully exposed parts especially in the N-terminal V domain.

post to: CiteULike

Solution Structure of the Heterotrimeric Complex between the Interaction Domains of RFX5 and RFXAP from the RFX Gene Regulatory Complex.

Publication Date: 2010 Aug 21 PMID: 20732328
Authors: Laird, K. M. - Briggs, L. L. - Boss, J. M. - Summers, M. F. - Garvie, C. W.
Journal: J Mol Biol

The mammalian immune response is mediated by a heterotetrameric transcriptional control complex, called regulatory factor X (RFX), that regulates the expression of major histocompatibility complex class II genes. RFX comprises four proteins: RFX5 (two copies), RFXAP, and RFXB, and mutations and deletions that prevent the assembly of the RFX complex have been linked to a severe immunodeficiency disorder. Two RFX5 molecules and one RFXAP molecule assemble in the cytoplasm prior to nuclear localization, a process mediated by an N-terminal "dimerization domain" of RFX5 (RFX5(N)) and a C-terminal domain of RFXAP (RFXAP(C)). We previously presented evidence that RFXAP(C) is unstructured in the absence of RFX5(N) but adopts a regular structure in the RFX5(N)(2)-RFXAP(C) complex and that the RFX5(N)(2)-RFXAP(C) complex binds RFXB with high affinity. We now report the structure of the RFX5(N)(2)-RFXAP(C) complex, determined in solution by (15)N- and (13)C-edited NMR spectroscopy. RFX5(N) consists of a long central helix flanked by two shorter helices. The central helices of the two RFX5(N) molecules form an antiparallel coiled coil, and the flanking helices pack at the ends of the long helices in a perpendicular arrangement such that the RFX5(N) dimer is shaped like a staple. RFXAP(C) consists of two alpha-helices that form a V-shaped structure that packs within the RFX5(N)(2) staple. Leucine residues in the leucine-rich region of RFX5(N) (62-LYLYLQL-68) that are critical for major histocompatibility complex class II gene expression in vivo contribute to both the dimer (Leu64 and Leu68) and the RFX5(N)-RFXAP(C) interfaces (Leu62 and Leu66). The clustering of hydrophobic residues from different regions of RFXAP(C) suggests a potential binding site for RFXB.

post to: CiteULike

Structural Analysis of the Interactions Between Hsp70 Chaperones and the Yeast DNA Replication Protein Orc4p.

Publication Date: 2010 Aug 21 PMID: 20732327
Authors: Alamo, M. M. - Sanchez-Gorostiaga, A. - Serrano, A. M. - Prieto, A. - Cuellar, J. - Martin-Benito, J. - Valpuesta, J. M. - Giraldo, R.
Journal: J Mol Biol

Hsp70 chaperones, besides their role in assisting protein folding, are key modulators of protein disaggregation, being consistently found as components of most macromolecular assemblies isolated in proteome-wide affinity purifications. A wealth of structural information has been recently acquired on Hsp70s complexed with Hsp40 and NEF co-factors and with small hydrophobic target peptides. However, knowledge of how Hsp70s recognize large protein substrates is still limited. Earlier, we reported that homologue Hsp70 chaperones (DnaK in Escherichia coli and Ssa1-4p/Ssb1-2p in Saccharomyces cerevisiae) bind strongly, both in vitro and in vivo, to the AAA+ domain in the Orc4p subunit of yeast origin recognition complex (ORC). ScORC is the paradigm for eukaryotic DNA replication initiators and consists of six distinct protein subunits (ScOrc1-p-ScOrc 6p). Here, we report that a hydrophobic sequence (IL(4)) in the initiator specific motif (ISM) in Orc4p is the main target for DnaK/Hsp70. The three-dimensional electron microscopy reconstruction of a stable Orc4p(2)-DnaK complex suggests that the C-terminal substrate-binding domain in the chaperone clamps the AAA+ IL(4) motif in one Orc4p molecule, with the substrate-binding domain lid subdomain wedging apart the other Orc4p subunit. Pairwise co-expression in E. coli shows that Orc4p interacts with Orc1/2/5p. Mutation of IL(4) selectively disrupts Orc4p interaction with Orc2p. Allelic substitution of ORC4 by mutants in each residue of IL(4) results in lethal (I184A) or thermosensitive (L185A and L186A) initiation-defective phenotypes in vivo. The interplay between Hsp70 chaperones and the Orc4p-IL(4) motif might have an adaptor role in the sequential, stoichiometric assembly of ScORC subunits.

post to: CiteULike

Molecular Insight into the Conformational Dynamics of the Elongin BC Complex and Its Interaction with HIV-1 Vif.

Publication Date: 2010 Aug 20 PMID: 20728451
Authors: Marcsisin, S. R. - Engen, J. R.
Journal: J Mol Biol

The human immunodeficiency virus type 1 virion infectivity factor (Vif) inhibits the innate viral immunity afforded by the APOBEC3 family of cytidine deaminases. Vif targets the APOBEC3 family for poly-ubiquitination and subsequent proteasomal degradation by linking the Elongin-BC-dependent ubiquitin ligase complex with the APOBEC3 proteins. The interaction between Vif and the heterodimeric Elongin BC complex, which is mediated by Vif's viral suppressor of cytokine signaling box, is essential for Vif function. The biophysical consequences of the full-length Vif:Elongin BC interaction have not been extensively reported. In this study, hydrogen exchange mass spectrometry was used to dissect the Vif:Elongin BC interaction. Elongin C was found to be highly dynamic in the Elongin BC complex while Elongin B was much more stable. Recombinant full-length Vif interacted with the Elongin BC complex in vitro with a K(d) of 1.9 muM and resulted in observable changes in deuterium uptake in both Elongin C and B. Upon binding to Elongin BC, no significant global conformational changes were detected in Vif by hydrogen exchange mass spectrometry, but a short fragment of Vif that consisted of the viral suppressor of cytokine signaling box showed decreased deuterium incorporation upon Elongin BC incubation, suggesting that this region folds upon binding.

post to: CiteULike

Crystal Structures of the Substrate-Bound Forms of Red Chlorophyll Catabolite Reductase: Implications for Site-Specific and Stereospecific Reaction.

Publication Date: 2010 Aug 19 PMID: 20727901
Authors: Sugishima, M. - Okamoto, Y. - Noguchi, M. - Kohchi, T. - Tamiaki, H. - Fukuyama, K.
Journal: J Mol Biol

Red chlorophyll catabolite reductase (RCCR) catalyzes the ferredoxin-dependent reduction of the C20/C1 double bond of red chlorophyll catabolite (RCC), the catabolic intermediate produced in chlorophyll degradation. The crystal structure of substrate-free Arabidopsis thaliana RCCR (AtRCCR) demonstrated that RCCR folds into a characteristic alpha/beta/alpha sandwich, similar to that observed in the ferredoxin-dependent bilin reductase (FDBR) family. Here we have determined the crystal structures of RCC-bound AtRCCR, RCC-bound F218 V AtRCCR, and substrate-free F218 V AtRCCR, a mutant protein that produces the stereoisomer of primary fluorescent chlorophyll catabolites at the C1 position. RCC is bound to the pocket between the beta-sheet and the C-terminal alpha-helices, as seen in substrate-bound FDBRs, but RCC binding to RCCR is much looser than substrate binding to FDBRs. The loose binding seems beneficial to the large conformational change in RCC upon reduction. Two conserved acidic residues, Glu154 and Asp291, sandwich the C20/C1 double bond of RCC, suggesting that these two residues are involved in site-specific reduction. The RCC in F218V AtRCCR rotates slightly compared with that in wild type to fill in the space generated by the substitution of Phe218 with valine. Concomitantly, the two carboxy groups of Glu154 and Asp291 move slightly away from the C20/C1 double bond. The geometrical arrangement of RCC and the carboxy groups of Glu154 and Asp291 in RCCR would appear to be essential for the stereospecificity of the RCCR reaction.

post to: CiteULike

Crystal Structure of Zebrafish Hatching Enzyme 1 from the Zebrafish Danio rerio.

Publication Date: 2010 Aug 18 PMID: 20727360
Authors: Okada, A. - Sano, K. - Nagata, K. - Yasumasu, S. - Ohtsuka, J. - Yamamura, A. - Kubota, K. - Iuchi, I. - Tanokura, M.
Journal: J Mol Biol

Fish hatching enzymes are zinc metalloproteases that digest the egg envelope (chorion) at the time of hatching. The crystal structure of zebrafish hatching enzyme 1 (ZHE1) has been solved at 1.10 A resolution. ZHE1 is monomeric, is mitten shaped, and has a cleft at the center of the molecule. ZHE1 consists of three 3(10)-helices, three alpha-helices, and two beta-sheets. The central cleft represents the active site of the enzyme that is crucial for substrate recognition and catalysis. Alanine-scanning mutagenesis of the two substrate peptides has shown that AspP1' contributes the most and that the residues at P4-P2' also contribute to the recognition of the major substrate peptide by ZHE1, whereas GluP3' and the hydrophobic residues at P4-P2, P2', and P5' contribute significantly to the recognition of the minor substrate peptide by ZHE1. Molecular models of these two substrate peptides bound to ZHE1 have been built based on the crystal structure of a transition-state analog inhibitor bound to astacin. In substrate-recognition models, the AspP1' in the major substrate peptide forms a salt bridge with Arg182 of ZHE1, while the GluP3' in the minor substrate peptide instead forms a salt bridge with Arg182. Thus, these two substrate peptides would be differently recognized by ZHE1. The shapes and electrostatic potentials of the substrate-binding clefts of ZHE1 and the structurally similar proteins astacin and bone morphogenetic protein 1 are significantly dissimilar due to different side chains, which would confer their distinctive substrate preferences.

post to: CiteULike

Coevolution of Antibody Stability and Vkappa CDR-L3 Canonical Structure.

Publication Date: 2010 Aug 18 PMID: 20727359
Authors: Luo, J. - Obmolova, G. - Huang, A. - Muzammil, S. - Teplyakov, A. - Malia, T. - Strake, B. - Zhao, Y. - Gilliland, G. L. - Feng, Y.
Journal: J Mol Biol

Antibodies recognize antigens through six hypervariable loops, five of which have a limited set of conformations known as canonical structures. For kappa light chains, the majority of CDR-L3 [the third hypervariable loop of the variable light domain (V(L))] adopts the type 1 canonical structure (CS1), with a cis-proline at position 95. Here, we present the design and structural studies of the monoclonal antibody mAb15 and related mutants that contained a series of progressively germ line mutations only in the variable heavy domain (V(H)) that ultimately led to an increase of more than 11 degrees C in antigen-binding fragment melting temperature (T(m)). The all-trans CDR-L3 structure in the wild type is significantly different from any known CDR-L3 canonical structures. In the thermally stable mutants, the L94(L)-S95(L) peptide bond adopts an energetically unfavorable non-X-proline cis conformation, but the overall CDR-L3 loop converted to CS1. The stabilized V(H) appears to function as a specific molecular chaperone that facilitated the trans-cis isomerization of S95(L). Thus, it is plausible that proline is the evolutionary choice to maintain overall structure and stability for V(L). These results provide new insights into the evolution of CS1 and suggest a potential molecular switch mechanism at position 95 that links CDR-L3 structural diversity and antibody stability and will have implications for antibody engineering.

post to: CiteULike

Multifactorial Determinants of Protein Expression in Prokaryotic Open Reading Frames.

Publication Date: 2010 Aug 18 PMID: 20727358
Authors: Allert, M. - Cox, J. C. - Hellinga, H. W.
Journal: J Mol Biol

A quantitative description of the relationship between protein expression levels and open reading frame (ORF) nucleotide sequences is important for understanding natural systems, designing synthetic systems, and optimizing heterologous expression. Codon identity, mRNA secondary structure, and nucleotide composition within ORFs markedly influence expression levels. Bioinformatic analysis of ORF sequences in 816 bacterial genomes revealed that these features show distinct regional trends. To investigate their effects on protein expression, we designed 285 synthetic genes and determined corresponding expression levels in vitro using Escherichia coli extracts. We developed a mathematical function, parameterized using this synthetic gene data set, which enables computation of protein expression levels from ORF nucleotide sequences. In addition to its practical application in the design of heterologous expression systems, this equation provides mechanistic insight into the factors that control translation efficiency. We found that expression is strongly dependent on the presence of high AU content and low secondary structure in the ORF 5' region. Choice of high-frequency codons contributes to a lesser extent. The 3' terminal AU content makes modest, but detectable contributions. We present a model for the effect of these factors on the three phases of ribosomal function: initiation, elongation, and termination.

post to: CiteULike

Magnesium-Dependent Interaction of PKR with Adenovirus VAI.

Publication Date: 2010 Aug 14 PMID: 20713064
Authors: Launer-Felty, K. - Wong, C. J. - Wahid, A. M. - Conn, G. L. - Cole, J. L.
Journal: J Mol Biol

Protein kinase R (PKR) is an interferon-induced kinase that plays a pivotal role in the innate immunity pathway for defense against viral infection. PKR is activated to undergo autophosphorylation upon binding to RNAs that contain duplex regions. Activated PKR phosphorylates the alpha-subunit of eukaryotic initiation factor 2, thereby inhibiting protein synthesis in virus-infected cells. Viruses have evolved diverse PKR-inhibitory strategies to evade antiviral response. Adenovirus encodes virus-associated RNA I (VAI), a highly structured RNA inhibitor that binds PKR but fails to activate. We have characterized the stoichiometry and affinity of PKR binding to define the mechanism of PKR inhibition by VAI. Sedimentation velocity and isothermal titration calorimetry measurements indicate that PKR interactions with VAI are modulated by Mg(2+). Two PKR monomers bind in the absence of Mg(2+), but a single monomer binds in the presence of divalent ion. Known RNA activators of PKR are capable of binding multiple PKR monomers to allow the kinase domains to come into close proximity and thus enhance dimerization. We propose that VAI acts as an inhibitor of PKR because it binds and sequesters a single PKR in the presence of divalent cation.

post to: CiteULike

Thermodynamic Characterization of ppGpp Binding to EF-G or IF2 and of Initiator tRNA Binding to Free IF2 in the Presence of GDP, GTP, or ppGpp.

Publication Date: 2010 Aug 14 PMID: 20713063
Authors: Mitkevich, V. A. - Ermakov, A. - Kulikova, A. A. - Tankov, S. - Shyp, V. - Soosaar, A. - Tenson, T. - Makarov, A. A. - Ehrenberg, M. - Hauryliuk, V.
Journal: J Mol Biol

In addition to their natural substrates GDP and GTP, the bacterial translational GTPases initiation factor (IF) 2 and elongation factor G (EF-G) interact with the alarmone molecule guanosine tetraphosphate (ppGpp), which leads to GTPase inhibition. We have used isothermal titration calorimetry to determine the affinities of ppGpp for IF2 and EF-G at a temperature interval of 5-25 degrees C. We find that ppGpp has a higher affinity for IF2 than for EF-G (1.7-2.8 muM K(d)versus 9.1-13.9 muM K(d) at 10-25 degrees C), suggesting that during stringent response in vivo, IF2 is more responsive to ppGpp than to EF-G. We investigated the effects of ppGpp, GDP, and GTP on IF2 interactions with fMet-tRNA(fMet) demonstrating that IF2 binds to initiator tRNA with submicromolar K(d) and that affinity is altered by the G nucleotides only slightly. This-in conjunction with earlier reports on IF2 interactions with fMet-tRNA(fMet) in the context of the 30S initiation complex, where ppGpp was suggested to strongly inhibit fMet-tRNA(fMet) binding and GTP was suggested to strongly promote fMet-tRNA(fMet) binding-sheds new light on the mechanisms of the G-nucleotide-regulated fMet-tRNA(fMet) selection.

post to: CiteULike

DNA Minor Groove Induced Dimerization of Heterocyclic Cations: Compound Structure, Binding Affinity, and Specificity for a TTAA Site.

Publication Date: 2010 Aug 14 PMID: 20713062
Authors: Munde, M. - Kumar, A. - Nhili, R. - Depauw, S. - David-Cordonnier, M. H. - Ismail, M. A. - Stephens, C. E. - Farahat, A. A. - Batista-Parra, A. - Boykin, D. W. - Wilson, W. D.
Journal: J Mol Biol

With the increasing number and variations of genome sequences available, control of gene expression with synthetic, cell-permeable molecules is within reach. The variety of sequence-specific binding agents is, however, still quite limited. Many minor groove binding agents selectivity recognize AT over GC sequences but have less ability to distinguish among different AT sequences. The goal with this article is to develop compounds that can bind selectively to different AT sequences. A number of studies indicate that AATT and TTAA sequences have significantly different physical and interaction properties and different requirements for minor groove recognition. Although it has been difficult to get minor groove binding at TTAA, DB293, a phenyl-furan-benzimidazole diamidine, was found to bind as a strong, cooperative dimer at TTAA but with no selectivity over AATT. In order to improve selectivity, we made modifications to each unit of DB293. Binding affinities and stoichiometries obtained from biosensor-surface plasmon resonance experiments show that DB1003, a furan-furan-benzimidazole diamidine, binds strongly to TTAA as a dimer and has selectivity (K(TTAA)/K(AATT)=6). CD and DNase I footprinting studies confirmed the preference of this compound for TTAA. In summary, (i) a favorable stacking surface provided by the pi system, (ii) H-bond donors to interact with TA base pairs at the floor of the groove provided by a benzimidazole (or indole) -NH and amidines, and (iii) appropriate curvature of the dimer complex to match the curvature of the minor groove play important roles in differentiating the TTAA and AATT minor grooves.

post to: CiteULike

Further Insight into Substrate Recognition by USP7: Structural and Biochemical Analysis of the HdmX and Hdm2 Interactions with USP7.

Publication Date: 2010 Aug 14 PMID: 20713061
Authors: Sarkari, F. - La Delfa, A. - Arrowsmith, C. H. - Frappier, L. - Sheng, Y. - Saridakis, V.
Journal: J Mol Biol

Ubiquitin-specific protease 7 (USP7) catalyzes the deubiquitination of several substrate proteins including p53 and Hdm2. We have previously shown that USP7, and more specifically its amino-terminal domain (USP7-NTD), interacts with distinct regions on p53 and Hdm2 containing P/AxxS motifs. The ability of USP7 to also deubiquitinate and control the turnover of HdmX was recently demonstrated. We utilized a combination of biochemistry and structural biology to identify which domain of USP7 interacts with HdmX as well as to identify regions of HdmX that interact with USP7. We showed that USP7-NTD recognized two of six P/AxxS motifs of HdmX ((8)AQCS(11) and (398)AHSS(401)). The crystal structure of the USP7-NTD:HdmX(AHSS) complex was determined providing the molecular basis of complex formation between USP7-NTD and the HdmX(AHSS) peptide. The HdmX peptide interacted within the same residues of USP7-NTD as previously demonstrated with p53, Hdm2, and EBNA1 peptides. We also identified an additional site on Hdm2 ((397)PSTS(400)) that interacts with USP7-NTD and determined the crystal structure of this complex. Finally, analysis of USP7-interacting peptides on filter arrays confirmed the importance of the serine residue at the fourth position for the USP7-NTD interaction and showed that phosphorylation of serines within the binding sequence prevents this interaction. These results lead to a better understanding of the mechanism of substrate recognition by USP7-NTD.

post to: CiteULike

Context-Dependent Remodeling of Structure in Two Large Protein Fragments.

Publication Date: 2010 Aug 14 PMID: 20713060
Authors: Schellenberg, M. J. - Ritchie, D. B. - Wu, T. - Markin, C. J. - Spyracopoulos, L. - Macmillan, A. M.
Journal: J Mol Biol

Protein folding involves the formation of secondary structural elements from the primary sequence and their association with tertiary assemblies. The relation of this primary sequence to a specific folded protein structure remains a central question in structural biology. An increasing body of evidence suggests that variations in homologous sequence ranging from point mutations to substantial insertions or deletions can yield stable proteins with markedly different folds. Here we report the structural characterization of domain IV (D4) and DeltaD4 (polypeptides with 222 and 160 amino acids, respectively) that differ by virtue of an N-terminal deletion of 62 amino acids (28% of the overall D4 sequence). The high-resolution crystal structures of the monomeric D4 and the dimeric DeltaD4 reveal substantially different folds despite an overall conservation of secondary structure. These structures show that the formation of tertiary structures, even in extended polypeptide sequences, can be highly context dependent, and they serve as a model for structural plasticity in protein isoforms.

post to: CiteULike

NMR Structure of the SARS-CoV Nonstructural Protein 7 in Solution at pH 6.5.

Publication Date: 2010 Aug 13 PMID: 20709084
Authors: Johnson, M. A. - Jaudzems, K. - Wuthrich, K.
Journal: J Mol Biol

The NMR structure of the severe acute respiratory syndrome coronavirus nonstructural protein (nsp) 7 in aqueous solution at pH 6.5 was determined and compared with the results of previous structure determinations of nsp7 in solution at pH 7.5 and in the crystals of a hexadecameric nsp7/nsp8 complex obtained from a solution at pH 7.5. All three structures contain four helices as the only regular secondary structures, but there are differences in the lengths and sequence locations of the four helices, as well as between the tertiary folds. The present study includes data on conformational equilibria and intramolecular rate processes in nsp7 in solution at pH 6.5, which provide further insights into the polymorphisms implicated by a comparison of the three presently available nsp7 structures.

post to: CiteULike

Influence of the Cytoplasmic Domains of Aquaporin-4 on Water Conduction and Array Formation.

Publication Date: 2010 Aug 13 PMID: 20709083
Authors: Mitsuma, T. - Tani, K. - Hiroaki, Y. - Kamegawa, A. - Suzuki, H. - Hibino, H. - Kurachi, Y. - Fujiyoshi, Y.
Journal: J Mol Biol

Phosphorylation of Ser180 in cytoplasmic loop D has been shown to reduce the water permeability of aquaporin (AQP) 4, the predominant water channel in the brain. However, when the structure of the S180D mutant (AQP4M23S180D), which was generated to mimic phosphorylated Ser180, was determined to 2.8 A resolution using electron diffraction patterns, it showed no significant differences from the structure of the wild-type channel. High-resolution density maps usually do not resolve protein regions that are only partially ordered, but these can sometimes be seen in lower-resolution density maps calculated from electron micrographs. We therefore used images of two-dimensional crystals and determined the structure of AQP4M23S180D at 10 A resolution. The features of the 10-A density map are consistent with those of the previously determined atomic model; in particular, there were no indications of any obstruction near the cytoplasmic pore entrance. In addition, water conductance measurements, both in vitro and in vivo, show the same water permeability for wild-type and mutant AQP4M23, suggesting that the S180D mutation neither reduces water conduction through a conformational change nor reduces water conduction by interacting with a protein that would obstruct the cytoplasmic channel entrance. Finally, the 10-A map shows a cytoplasmic density in between four adjacent tetramers that most likely represents the association of four N termini. This finding supports the critical role of the N terminus of AQP4 in the stabilization of orthogonal arrays, as well as their interference through lipid modification of cysteine residues in the longer N-terminal isoform.

post to: CiteULike

Visualizing the Structural Changes of Bacteriophage Epsilon15 and Its Salmonella Host during Infection.

Publication Date: 2010 Aug 13 PMID: 20709082
Authors: Chang, J. T. - Schmid, M. F. - Haase-Pettingell, C. - Weigele, P. R. - King, J. A. - Chiu, W.
Journal: J Mol Biol

The efficient mechanism by which double-stranded DNA bacteriophages deliver their chromosome across the outer membrane, cell wall, and inner membrane of Gram-negative bacteria remains obscure. Advances in single-particle electron cryomicroscopy have recently revealed details of the organization of the DNA injection apparatus within the mature virion for various bacteriophages, including epsilon15 (varepsilon15) and P-SSP7. We have used electron cryotomography and three-dimensional subvolume averaging to capture snapshots of varepsilon15 infecting its host Salmonella anatum. These structures suggest the following stages of infection. In the first stage, the tailspikes of varepsilon15 attach to the surface of the host cell. Next, varepsilon15's tail hub attaches to a putative cell receptor and establishes a tunnel through which the injection core proteins behind the portal exit the virion. A tube spanning the periplasmic space is formed for viral DNA passage, presumably from the rearrangement of core proteins or from cellular components. This tube would direct the DNA into the cytoplasm and protect it from periplasmic nucleases. Once the DNA has been injected into the cell, the tube and portal seals, and the empty bacteriophage remains at the cell surface.

post to: CiteULike

Micelle-Like Architecture of the Monomer Ensemble of Alzheimer's Amyloid-beta Peptide in Aqueous Solution and Its Implications for Abeta Aggregation.

Publication Date: 2010 Aug 13 PMID: 20709081
Authors: Vitalis, A. - Caflisch, A.
Journal: J Mol Biol

Aggregation of amyloid-beta (Abeta) peptide, a 39-residue to 43-residue fragment of the amyloid precursor protein, is associated with Alzheimer's disease, the most common form of dementia in the elderly population. Several experimental studies have tried to characterize the atomic details of amyloid fibrils, which are the final product of Abeta aggregation. Much less is known about species forming during the early stages of aggregation, in particular about the monomeric state of the Abeta peptide (i.e., the product of the very first step in the amyloid cascade hypothesis). Here, the equilibrium ensembles of the monomeric Abeta alloforms Abeta(1-40) and Abeta(1-42) are investigated by Monte Carlo simulations using an atomistic force field and an implicit solvent model that have been shown previously to correctly reproduce the ensemble properties of other intrinsically disordered polypeptides. Simulation results indicate that at physiological temperatures, both alloforms of Abeta assume a largely collapsed globular structure, with a fluid hydrophobic core (formed, on average, by contacts both within and between segments) comprising residues 12-21 and 24-40/42. Furthermore, the 11 N-terminal residues are completely unstructured, and all charged side chains, in particular those of Glu22 and Asp23, remain exposed to solvent. Taken together, these observations indicate a micelle-like (The terms "micelle-like architecture" and "micelle-like" used throughout this article refer to the average conformation of an individual polypeptide chain and do not imply the assembly of several molecules, unless otherwise noted.) architecture at the monomer level whose implications for oligomerization, as well as fibril formation and elongation, are discussed. We establish quantitatively the intrinsic disorder of Abeta and find the propensity to form a regular secondary structure to be low but sequence specific. In the presence of a global and unspecific bias for backbone conformations to populate the beta-basin, the beta-sheet propensity along the sequence is consistent with the arrangement of the monomer within the fibril, as derived from solid-state NMR data. These observations indicate that the primary sequence partially encodes fibril structure, but that fibril elongation must be thought of as a templated assembly step.

post to: CiteULike

SAXS and X-ray Crystallography Suggest an Unfolding Model for the GDP/GTP Conformational Switch of the Small GTPase Arf6.

Publication Date: 2010 Aug 13 PMID: 20709080
Authors: Biou, V. - Aizel, K. - Roblin, P. - Thureau, A. - Jacquet, E. - Hansson, S. - Guibert, B. - Guittet, E. - van Heijenoort, C. - Zeghouf, M. - Perez, J. - Cherfils, J.
Journal: J Mol Biol

The small GTPases Arf1 and Arf6 have nonoverlapping functions in cellular traffic despite their very high sequence and structural resemblance. Notably, the exquisite isoform specificity of their guanine nucleotide exchange factors and their distinctive sensitivity to the drug brefeldin A cannot be explained by any straightforward structural model. Here we integrated structural and spectroscopic methods to address this issue using Delta13Arf6-GDP, a truncated mutant that mimics membrane-bound Arf6-GDP. The crystal structure of Delta13Arf6-GDP reveals an unprecedented unfolding of the GTPase core beta-strands, which is fully accounted for by small-angle X-ray scattering data in solution and by ab initio three-dimensional envelope calculation. NMR chemical shifts identify this structural disorder in Delta13Arf6-GDP, but not in the closely related Delta17Arf1-GDP, which is consistent with their comparative thermodynamic and hydrodynamic analyses. Taken together, these experiments suggest an unfolding model for the nucleotide switch of Arf6 and shed new light on its biochemical differences with Arf1.

post to: CiteULike

Quantitative Proteomic Analysis of Ribosome Assembly and Turnover In Vivo.

Publication Date: 2010 Aug 13 PMID: 20709079
Authors: Sykes, M. T. - Shajani, Z. - Sperling, E. - Beck, A. H. - Williamson, J. R.
Journal: J Mol Biol

Although high-resolution structures of the ribosome have been solved in a series of functional states, relatively little is known about how the ribosome assembles, particularly in vivo. Here, a general method is presented for studying the dynamics of ribosome assembly and ribosomal assembly intermediates. Since significant quantities of assembly intermediates are not present under normal growth conditions, the antibiotic neomycin is used to perturb wild-type Escherichia coli. Treatment of E. coli with the antibiotic neomycin results in the accumulation of a continuum of assembly intermediates for both the 30S and 50S subunits. The protein composition and the protein stoichiometry of these intermediates were determined by quantitative mass spectrometry using purified unlabeled and (15)N-labeled wild-type ribosomes as external standards. The intermediates throughout the continuum are heterogeneous and are largely depleted of late-binding proteins. Pulse labeling with (15)N-labeled medium time-stamps the ribosomal proteins based on their time of synthesis. The assembly intermediates contain both newly synthesized proteins and proteins that originated in previously synthesized intact subunits. This observation requires either a significant amount of ribosome degradation or the exchange or reuse of ribosomal proteins. These specific methods can be applied to any system where ribosomal assembly intermediates accumulate, including strains with deletions or mutations of assembly factors. This general approach can be applied to study the dynamics of assembly and turnover of other macromolecular complexes that can be isolated from cells.

post to: CiteULike

Proteome-Level Relationship between Folding and Aggregation Propensities of Proteins.

Publication Date: 2010 Aug 13 PMID: 20709078
Authors: Tartaglia, G. G. - Vendruscolo, M.
Journal: J Mol Biol

With the advent of proteomics, there is an increasing need of tools for predicting the properties of large numbers of proteins by using the information provided by their amino acid sequences, even in the absence of the knowledge of their structures. One of the most important types of predictions concerns whether proteins will fold or aggregate. Here, we study the competition between these two processes by analyzing the relationship between the folding and the aggregation propensity profiles for the human and Escherichia coli proteomes. These profiles are calculated, respectively, using the CamFold method, which we introduce in this work, and the Zyggregator method. Our results indicate that the kinetic behavior of proteins is, to a large extent, determined by the interplay between regions of low folding and high aggregation propensities.

post to: CiteULike

Modeling Studies of Chromatin Fiber Structure as a Function of DNA Linker Length.

Publication Date: 2010 Aug 13 PMID: 20709077
Authors: Perisic, O. - Collepardo-Guevara, R. - Schlick, T.
Journal: J Mol Biol

Chromatin fibers encountered in various species and tissues are characterized by different nucleosome repeat lengths (NRLs) of the linker DNA connecting the nucleosomes. While single cellular organisms and rapidly growing cells with high protein production have short NRL ranging from 160 to 189 bp, mature cells usually have longer NRLs ranging between 190 and 220 bp. Recently, various experimental studies have examined the effect of NRL on the internal organization of chromatin fiber. Here, we investigate by mesoscale modeling of oligonucleosomes the folding patterns for different NRL, with and without linker histone (LH), under typical monovalent salt conditions using both one-start solenoid and two-start zigzag starting configurations. We find that short to medium NRL chromatin fibers (173 to 209 bp) with LH condense into irregular zigzag structures and that solenoid-like features are viable only for longer NRLs (226 bp). We suggest that medium NRLs are more advantageous for packing and various levels of chromatin compaction throughout the cell cycle than their shortest and longest brethren; the former (short NRLs) fold into narrow fibers, while the latter (long NRLs) arrays do not easily lead to high packing ratios due to possible linker DNA bending. Moreover, we show that the LH has a small effect on the condensation of short-NRL arrays but has an important condensation effect on medium-NRL arrays, which have linker lengths similar to the LH lengths. Finally, we suggest that the medium-NRL species, with densely packed fiber arrangements, may be advantageous for epigenetic control because their histone tail modifications can have a greater effect compared to other fibers due to their more extensive nucleosome interaction network.

post to: CiteULike

Unstructured Hydrophilic Sequences in Prokaryotic Proteomes Correlate with Dehydration Tolerance and Host Association.

Publication Date: 2010 Aug 13 PMID: 20709076
Authors: Krisko, A. - Smole, Z. - Debret, G. - Nikolic, N. - Radman, M.
Journal: J Mol Biol

Water loss or desiccation is among the most life-threatening stresses. It leads to DNA double-strand breakage, protein aggregation, cell shrinkage, and low water activity precluding all biological functions. Yet, in all kingdoms of life, rare organisms are resistant to desiccation through prevention or reversibility of such damage. Here, we explore possible hallmarks of prokaryotic desiccation tolerance in their proteomes. The content of unstructured, low complexity (LC) regions was analyzed in a total of 460 bacterial and archaeal proteomes. It appears that species endowed with proteomes abundant in unstructured hydrophilic LC regions are desiccation-tolerant or sporulating bacteria, halophilic archaea and bacteria, or host-associated species. In the desiccation- and radiation-resistant bacterium Deinococcus radiodurans, most proteins that contain large hydrophilic LC regions have unassigned function, but those with known function are mostly involved in diverse cellular recovery processes. Such LC regions are typically absent in orthologous proteins in desiccation-sensitive species. D. radiodurans encodes also special LC proteins, akin to those associated with desiccation resistance of plant seeds and some plants and animals. Therefore, we postulate that large unstructured hydrophilic LC regions and proteins provide for cellular resistance to dehydration and we discuss mechanisms of their protective activity.

post to: CiteULike

Global Network Analysis of Lipid-Raft-Related Proteins Reveals Their Centrality in the Network and Their Roles in Multiple Biological Processes.

Publication Date: 2010 Aug 13 PMID: 20709075
Authors: Zhang, T. - Zhang, X. - Sun, Z.
Journal: J Mol Biol

Lipid rafts are specialized cholesterol-enriched microdomains in the cell membrane. They have been known as a platform for protein-protein interactions and to take part in multiple biological processes. Nevertheless, how lipid rafts influence protein properties at the proteomic level is still an open question for researchers using traditional biochemical approaches. Here, by annotating the lipid raft localization of proteins in human protein-protein interaction networks, we performed a systematic analysis of the function of proteins related to lipid rafts. Our results demonstrated that lipid raft proteins and their interactions were critical for the structure and stability of the whole network, and that the interactions between them were significantly enriched. Furthermore, for each protein in the network, we calculated for "lipid raft dependency (LRD)," which indicates how close it is topologically associated with lipid rafts, and we then uncovered the connection between LRD and protein functions. Proteins with high LRD tended to be essential for mammalian development, and malfunction of these proteins was inclined to cause human diseases. Coordinated with their neighbors, high-LRD proteins participated in multiple biological processes and targeted many pathways in diseases pathogenesis. High-LRD proteins were also found to have tissue specificity of expression. In summary, our network-based analysis denotes that lipid raft proteins have higher centrality in the network, and that lipid-raft-related proteins have multiple functions and are probably concerned with many biological processes in disease development.

post to: CiteULike

Dissecting the Microscopic Steps of the Cyclophilin A Enzymatic Cycle on the Biological Substrate HIV Capsid by NMR.

Publication Date: 2010 Aug 12 PMID: 20708627
Authors: Bosco, D. A. - Eisenmesser, E. Z. - Clarkson, M. W. - Wolf-Watz, M. - Labeikovsky, W. - Millet, O. - Kern, D.
Journal: J Mol Biol

Peptidyl-prolyl isomerases (PPIases) are emerging as key regulators of many diverse biological processes. Elucidating the role of PPIase activity in vivo has been challenging because mutagenesis of active-site residues not only reduces the catalytic activity of these enzymes but also dramatically affects substrate binding. Employing the cyclophilin A PPIase together with its biologically relevant and natively folded substrate, the N-terminal domain of the human immunodeficiency virus type 1 capsid (CA(N)) protein, we demonstrate here how to dissect residue-specific contributions to PPIase catalysis versus substrate binding utilizing NMR spectroscopy. Surprisingly, a number of cyclophilin A active-site mutants previously assumed to be strongly diminished in activity toward biological substrates based only on a peptide assay catalyze the human immunodeficiency virus capsid with wild-type activity but with a change in the rate-limiting step of the enzymatic cycle. The results illustrate that a quantitative analysis of catalysis using the biological substrates is critical when interpreting the effects of PPIase mutations in biological assays.

post to: CiteULike

H-Bonding and Positive Charge at the N(5)/O(4) Locus Are Critical for Covalent Flavin Attachment in Trametes Pyranose 2-Oxidase.

Publication Date: 2010 Aug 12 PMID: 20708626
Authors: Tan, T. C. - Pitsawong, W. - Wongnate, T. - Spadiut, O. - Haltrich, D. - Chaiyen, P. - Divne, C.
Journal: J Mol Biol

Flavoenzymes perform a wide range of redox reactions in nature, and a subclass of flavoenzymes carry covalently bound cofactor. The enzyme-flavin bond helps to increase the flavin's redox potential to facilitate substrate oxidation in several oxidases. The formation of the enzyme-flavin covalent bond-the flavinylation reaction-has been studied for the past 40 years. For the most advocated mechanism of autocatalytic flavinylation, the quinone methide mechanism, appropriate stabilization of developing negative charges at the flavin N(1) and N(5) loci is crucial. Whereas the structural basis for stabilization at N(1) is relatively well studied, the structural requisites for charge stabilization at N(5) remain less clear. Here, we show that flavinylation of histidine 167 of pyranose 2-oxidase from Trametes multicolor requires hydrogen bonding at the flavin N(5)/O(4) locus, which is offered by the side chain of Thr169 when the enzyme is in its closed, but not open, state. Moreover, our data show that additional stabilization at N(5) by histidine 548 is required to ensure high occupancy of the histidyl-flavin bond. The combination of structural and spectral data on pyranose 2-oxidase mutants supports the quinone methide mechanism. Our results demonstrate an elaborate structural fine-tuning of the active site to complete its own formation that couples efficient holoenzyme synthesis to conformational substates of the substrate-recognition loop and concerted movements of side chains near the flavinylation ligand.

post to: CiteULike

Crystal Structure of the Cyanobacterial Signal Transduction Protein P(II) in Complex with PipX.

Publication Date: 2010 Aug 12 PMID: 20708625
Authors: Zhao, M. X. - Jiang, Y. L. - Xu, B. Y. - Chen, Y. - Zhang, C. C. - Zhou, C. Z.
Journal: J Mol Biol

P(II) proteins are highly conserved signal transducers in bacteria, archaea, and plants. They have a large flexible loop (T-loop) that adopts different conformations after covalent modification or binding to different effectors to regulate the functions of diverse protein partners. The P(II) partner PipX (P(II)interaction protein X), first identified from Synechococcus sp. PCC 7942, exists uniquely in cyanobacteria. PipX also interacts with the cyanobacterial global nitrogen regulator NtcA. The mutually exclusive binding of P(II) and NtcA by PipX in a 2-oxoglutarate (2-OG)-dependent manner enables P(II) to indirectly regulate the transcriptional activity of NtcA. However, the structural basis for these exclusive interactions remains unknown. We solved the crystal structure of the P(II)-PipX complex from the filamentous cyanobacterium Anabaena sp. PCC 7120 at 1.90 A resolution. A homotrimeric P(II) captures three subunits of PipX through the T-loops. Similar to P(II) from Synechococcus, the core structure consists of an antiparallel beta-sheet with four beta-strands and two alpha-helices at the lateral surface. PipX adopts a novel structure composed of five twisted antiparallel beta-strands and two alpha-helices, which is reminiscent of the P(II) structure. The T-loop of each P(II) subunit extends from the core structure as an antenna that is stabilized at the cleft between two PipX monomers via hydrogen bonds. In addition, the interfaces between the beta-sheets of PipX and P(II) core structures partially contribute to complex formation. Comparative structural analysis indicated that PipX and 2-OG share a common binding site that overlaps with the 14 signature residues of cyanobacterial P(II) proteins. Our structure of PipX and the recently solved NtcA structure enabled us to propose a putative model for the NtcA-PipX complex. Taken together, these findings provide structural insights into how P(II) regulates the transcriptional activity of NtcA via PipX upon accumulation of the metabolite 2-OG.

post to: CiteULike

Structure of the AML1-ETO NHR3-PKA(RIIalpha) Complex and Its Contribution to AML1-ETO Activity.

Publication Date: 2010 Aug 11 PMID: 20708017
Authors: Corpora, T. - Roudaia, L. - Oo, Z. M. - Chen, W. - Manuylova, E. - Cai, X. - Chen, M. J. - Cierpecki, T. - Speck, N. A. - Bushweller, J. H.
Journal: J Mol Biol

AML1-ETO is the chimeric protein product of t(8;21) in acute myeloid leukemia. The ETO portion of the fusion protein includes the nervy homology region (NHR) 3 domain, which shares homology with A-kinase anchoring proteins and interacts with the regulatory subunit of type II cAMP-dependent protein kinase A (PKA(RIIalpha)). We determined the solution structure of a complex between the AML1-ETO NHR3 domain and PKA(RIIalpha). Based on this structure, a key residue in AML1-ETO for PKA(RIIalpha) association was mutated. This mutation did not disrupt AML1-ETO's ability to enhance the clonogenic capacity of primary mouse bone marrow cells or its ability to repress proliferation or granulocyte differentiation. Introduction of the mutation into AML1-ETO had minimal impact on in vivo leukemogenesis. Therefore, the NHR3-PKA(RIIalpha) protein interaction does not appear to significantly contribute to AML1-ETO's ability to induce leukemia.

post to: CiteULike

Conformation Changes in E. coli Rho Monitored by Hydrogen/Deuterium Exchange and Mass Spectrometry: Response to Ligand Binding.

Publication Date: 2010 Aug 11 PMID: 20708016
Authors: Stitt, B. L. - Xiao, H.
Journal: J Mol Biol

Escherichia coli Rho is a doughnut-shaped homohexameric ATP-dependent RNA-DNA helicase that releases newly synthesized RNA molecules from transcription complexes. Rho binds 60-80 bases of RNA among six primary RNA binding sites around the inside of its N-terminal crown; the RNA then passes through the central hole of the hexamer. Here it triggers ATP hydrolysis and is moved with respect to the protein. We study protein conformation changes upon ligand binding using amide proton hydrogen/deuterium exchange and mass spectrometry. Global-exchange studies indicate net mass differences of about 15 Da after 1 h of exchange in the presence-versus in the absence-of the ligand MgATP or the RNA poly(C). Sites of ligand-dependent exchange differences were localized by mass determination of the peptic peptides of Rho. A peptide of the N-terminal domain near the known primary RNA sites (aa 56-63) was protected from amide proton exchange in the presence of poly(C), as was a novel N-terminal domain peptide that is not near RNA in the crystal structures or in NMR structures with RNA oligomers (aa 37-46). This result may further define the primary interaction site of RNA with Rho. The Q-loop-containing peptide in the central hole of the protein that interacts with RNA was also protected by RNA (aa 271-286). The exchange rate of one peptide near the ATPase active site (aa 206-218) slowed in the presence of MgATP and increased in the presence of RNA. Overall, the results show changes in a few protein segments rather than a different overall conformation.

post to: CiteULike

Human RegIV Protein Adopts a Typical C-Type Lectin Fold but Binds Mannan with Two Calcium-Independent Sites.

Publication Date: 2010 Aug 6 PMID: 20692269
Authors: Ho, M. R. - Lou, Y. C. - Wei, S. Y. - Luo, S. C. - Lin, W. C. - Lyu, P. C. - Chen, C.
Journal: J Mol Biol

Human RegIV protein, which contains a sequence motif homologous to calcium-dependent (C-type) lectin-like domain, is highly expressed in mucosa cells of the gastrointestinal tract during pathogen infection and carcinogenesis and may be applied in both diagnosis and treatment of gastric and colon cancers. Here, we provide evidence that, unlike other C-type lectins, human RegIV binds to polysaccharides, mannan, and heparin in the absence of calcium. To elucidate the structural basis for carbohydrate recognition by NMR, we generated the mutant with Pro91 replaced by Ser (hRegIV-P91S) and showed that the structural property and carbohydrate binding ability of hRegIV-P91S are almost identical with those of wild-type protein. The solution structure of hRegIV-P91S was determined, showing that it adopts a typical fold of C-type lectin. Based on the chemical shift perturbations of amide resonances, two calcium-independent mannan-binding sites were proposed. One site is similar to the calcium-independent sugar-binding site on human RegIII and Langerin. Interestingly, the other site is adjacent to the conserved calcium-dependent site at position Ca-2 of typical C-type lectins. Moreover, model-free analysis of (15)N relaxation parameters and simplified Carr-Purcell-Meiboom-Gill relaxation dispersion experiments showed that a slow microsecond-to-millisecond time-scale backbone motion is involved in mannan binding by this site, suggesting a potential role for specific carbohydrate recognition. Our findings shed light on the sugar-binding mode of Reg family proteins, and we postulate that Reg family proteins evolved to bind sugar without calcium to keep the carbohydrate recognition activity under low-pH environments in the gastrointestinal tract.

post to: CiteULike

Crystal Structures of the Multidrug Binding Repressor Corynebacteriumglutamicum CgmR in Complex with Inducers and with an Operator.

Publication Date: 2010 Aug 5 PMID: 20691702
Authors: Itou, H. - Watanabe, N. - Yao, M. - Shirakihara, Y. - Tanaka, I.
Journal: J Mol Biol

CgmR (CGL2612) from Corynebacterium glutamicum is a multidrug-resistance-related transcription factor belonging to the TetR family, which is a protein family of widespread bacterial transcription factors typically involved in environmental response. Here, we report the crystal structures of CgmR homodimeric repressor in complex with two distinct inducers (1.95 and 1.4 A resolution) and with an operator (2.5 A resolution). The CgmR-operator complex showed that two CgmR dimers bound to the operator, and each half-site of the palindromic operator was asymmetrically recognized by two DNA-binding domains from different dimers on the opposite sides of the DNA. The inducer complexes demonstrated that both bound inducers act as a wedge to alter the operator-binding conformation of the repressor by steric inhibition. As steric hindrance is used, various drugs should act as inducers if they have sufficient volume for the conformation change and if their bindings sufficiently reduce free energy. The comparative structural study of CgmR free protein, in complex with operator, and with inducers, implies the other mechanism that might contribute to multidrug response of the repressor.

post to: CiteULike

Measuring "Unmeasurable" Folding Kinetics of Proteins by Single-Molecule Force Spectroscopy.

Publication Date: 2010 Aug 5 PMID: 20691701
Authors: Jollymore, A. - Li, H.
Journal: J Mol Biol

Folding and unfolding are fundamental biological processes in cell and are important for the biological functions of proteins. Characterizing the folding and unfolding kinetics of proteins is important for understanding the energetic landscape leading to the active native conformations of these molecules. However, the thermal or chemical-induced unfolding of many proteins is irreversible in vitro, precluding characterization of the folding kinetics of such proteins, just as it is impossible to "un-boil" an egg. Irreversible unfolding often manifests as irreversible aggregation of unfolded polypeptide chains, which typically occurs between denatured protein molecules in response to the exposure of hydrophobic residues to solvent. An example of such a protein where thermal denaturation results in irreversible aggregation is the beta-1,4 endoxylanase from Bacillus circulans (BCX). Here, we report the use of single-molecule atomic force microscopy to directly measure the folding kinetics of BCX in vitro. By mechanically unfolding BCX, we essentially allowed only one unfolded molecule to exist in solution at a given time, effectively eliminating the possibility for aggregation. We found that BCX can readily refold back to the native state, allowing us to measure its folding kinetics for the first time. Our results demonstrate that single-molecule force-spectroscopy-based methods can adequately tackle the challenge of "un-boiling eggs", providing a general methodology to characterize the folding kinetics of many proteins that suffer from irreversible denaturation and thus cannot be characterized using traditional equilibrium methodologies.

post to: CiteULike

A Structural Model for the DEAD Box Helicase YxiN in Solution: Localization of the RNA Binding Domain.

Publication Date: 2010 Aug 5 PMID: 20691700
Authors: Karow, A. R. - Klostermeier, D.
Journal: J Mol Biol

DEAD box proteins consist of a common helicase core formed by two globular RecA domains that are separated by a cleft. The helicase core acts as a nucleotide-dependent switch that alternates between open and closed conformations during the catalytic cycle of duplex separation, thereby providing basic helicase activity. Flanking domains can direct the helicase core to a specific RNA substrate by mediating high-affinity or high-specificity RNA binding. In addition, they may position RNA for the helicase core or may directly contribute to unwinding. While structures of different helicase cores have been determined previously, little is known about the orientation of flanking domains relative to the helicase core. YxiN is a DEAD box protein that consists of a helicase core and a C-terminal RNA binding domain (RBD) that mediates specific binding to hairpin 92 in 23S rRNA. To provide a framework for understanding the functional cooperation of the YxiN helicase core and the RBD, we mapped the orientation of the RBD in single-molecule fluorescence resonance energy transfer experiments. We present a model for the global conformation of YxiN in which the RBD lies above a slightly concave patch that is formed by flexible loops on the surface of the C-terminal RecA domain. The orientation of the RBD is different from the orientations of flanking domains in the Thermus thermophilus DEAD box protein Hera and in Saccharomyces cerevisiae Mss116p, in line with the different functions of these DEAD box proteins and of their RBDs. Interestingly, the corresponding patch on the C-terminal RecA domain that is covered by the YxiN RBD is also part of the interface between the translation factors eIF4A and eIF4G. Possibly, this region constitutes an adaptable interface that generally allows for the interaction of the helicase core with additional domains or interacting factors.

post to: CiteULike

The Role of L1 Stalk-tRNA Interaction in the Ribosome Elongation Cycle.

Publication Date: 2010 Aug 5 PMID: 20691699
Authors: Trabuco, L. G. - Schreiner, E. - Eargle, J. - Cornish, P. - Ha, T. - Luthey-Schulten, Z. - Schulten, K.
Journal: J Mol Biol

The ribosomal L1 stalk is a mobile structure implicated in directing tRNA movement during translocation through the ribosome. This article investigates three aspects of L1 stalk-tRNA interaction. First, by combining data from cryo electron microscopy, X-ray crystallography, and molecular dynamics simulations through the molecular dynamics flexible fitting method, we obtained atomic models of different tRNAs occupying the hybrid P/E state interacting with the L1 stalk. These models confirm the assignment of fluorescence resonance energy transfer states from previous single-molecule investigations of L1 stalk dynamics. Second, the models reconcile how initiator tRNA(fMet) interacts less strongly with the L1 stalk compared to elongator tRNA(Phe), as seen in previous single-molecule experiments. Third, results from a simulation of the entire ribosome in which the L1 stalk is moved from a half-closed conformation to its open conformation are found to support the hypothesis that L1 stalk opening is involved in tRNA release from the ribosome.

post to: CiteULike

Tail-Mediated Collapse of HMGB1 Is Dynamic and Occurs via Differential Binding of the Acidic Tail to the A and B Domains.

Publication Date: 2010 Aug 4 PMID: 20691192
Authors: Stott, K. - Watson, M. - Howe, F. S. - Grossmann, J. G. - Thomas, J. O.
Journal: J Mol Biol

The architectural DNA-binding protein HMGB1 consists of two tandem HMG-box domains joined by a basic linker to a C-terminal acidic tail, which negatively regulates HMGB1-DNA interactions by binding intramolecularly to the DNA-binding faces of both basic HMG boxes. Here we demonstrate, using NMR chemical-shift mapping at different salt concentrations, that the tail has a higher affinity for the B box and that A box-tail interactions are preferentially disrupted. Previously, we proposed a model in which the boxes are brought together in a collapsed, tail-mediated assembly, which is in dynamic equilibrium with a more extended form. Small-angle X-ray scattering data are consistent with such a dynamic equilibrium between collapsed and extended structures and are best represented by an ensemble. The ensembles contain a significantly higher proportion of collapsed structures when the tail is present. (15)N NMR relaxation measurements show that full-length HMGB1 has a significantly lower rate of rotational diffusion than the tail-less protein, consistent with the loss of independent domain motions in an assembled complex. Mapping studies using the paramagnetic spin label MTSL [(1-oxyl-2,2,5,5-tetramethyl-3-pyrrolidin-3-yl) methyl methanethiosulfonate] placed at three locations in the tail corroborate our previous findings that the tail binds to both boxes with some degree of specificity. The end of the tail lies further from the body of the protein and is therefore potentially free to interact with other proteins. MTSL labelling at a single site in the A domain (C44) causes detectable relaxation enhancements to B domain residues, suggesting the existence of a sandwich-like collapsed structure in which the tail enables the close approach of the basic domains. These intramolecular interactions are presumably important for the dynamic association of HMGB1 with chromatin and provide a mechanism by which protein-protein interactions or posttranslational modifications might regulate the protein's function at specific sites or stages in the cell cycle.

post to: CiteULike

Refolding and Polymerization Pathways of Neuroserpin.

Publication Date: 2010 Aug 4 PMID: 20691191
Authors: Takehara, S. - Zhang, J. - Yang, X. - Takahashi, N. - Mikami, B. - Onda, M.
Journal: J Mol Biol

Neuroserpin is a member of the serpin superfamily, and its mutants are retained within the endoplasmic reticulum of neurons as ordered polymers in association with dementia. It has been proposed that neuroserpin polymers are formed by a conformational change in the folded protein. However, an alternative model whereby polymers are formed during protein folding rather than from the folded protein has recently been proposed. We investigated the refolding and polymerization pathways of wild-type neuroserpin (WT) and of the pathogenic mutants S49P and H338R. Upon refolding, denatured WT immediately formed an initial refolding intermediate I(IN) and then underwent further refolding to the native form through a late refolding intermediate, I(R). The late-onset mutant S49P was also able to refold to the native form through I(IN) and I(R), but the final refolding step proceeded at a slower rate and with a lower refolding yield as compared with WT. The early-onset mutant H338R formed I(R) through the same pathway as S49P, but the protein could not attain the native state and remained as I(R). The I(R)s of the mutants had a long lifespan at 4 degrees C and thus were purified and characterized. Strikingly, when incubated under physiological conditions, I(R) formed ordered polymers with essentially the same properties as the polymers formed from the native protein. The results show that the mutants have a greater tendency to form polymers during protein folding than to form polymers from the folded protein. Our finding provides insights into biochemical approaches to treating serpinopathies by targeting a polymerogenic folding intermediate.

post to: CiteULike

Structural Basis for the Dual Recognition of IL-12 and IL-23 by Ustekinumab.

Publication Date: 2010 Aug 4 PMID: 20691190
Authors: Luo, J. - Wu, S. J. - Lacy, E. R. - Orlovsky, Y. - Baker, A. - Teplyakov, A. - Obmolova, G. - Heavner, G. - Richter, H. T. - Benson, J.
Journal: J Mol Biol

Interleukin (IL)-12 and IL-23 are heterodimeric proinflammatory cytokines that share a common p40 subunit, paired with p35 and p19 subunits, respectively. They represent an attractive class of therapeutic targets for the treatment of psoriasis and other immune-mediated diseases. Ustekinumab is a fully human monoclonal antibody (mAb) that binds specifically to IL-12/IL-23p40 and neutralizes human IL-12 and IL-23 bioactivity. The crystal structure of ustekinumab Fab (antigen binding fragment of mAb), in complex with human IL-12, has been determined by X-ray crystallography at 3.0 A resolution. Ustekinumab Fab binds the D1 domain of the p40 subunit in a 1:1 ratio in the crystal, consistent with a 2 cytokines:1 mAb stoichiometry, as measured by isothermal titration calorimetry. The structure indicates that ustekinumab binds to the same epitope on p40 in both IL-12 and IL-23 with identical interactions. Mutational analyses confirm that several residues identified in the IL-12/IL-23p40 epitope provide important molecular binding interactions with ustekinumab. The electrostatic complementarity between the mAb antigen binding site and the p40 D1 domain epitope appears to play a key role in antibody/antigen recognition specificity. Interestingly, this structure also reveals significant structural differences in the p35 subunit and p35/p40 interface, compared with the published crystal structure of human IL-12, suggesting an unusual and potentially functionally relevant structural flexibility of p35, as well as p40/p35 recognition. Collectively, these data describe unique observations about IL-12p35 and ustekinumab interactions with p40 that account for its dual binding and neutralization of IL-12 and IL-23.

post to: CiteULike

Structural Insight into the Mechanism of c-di-GMP Hydrolysis by EAL Domain Phosphodiesterases.

Publication Date: 2010 Aug 4 PMID: 20691189
Authors: Tchigvintsev, A. - Xu, X. - Singer, A. - Chang, C. - Brown, G. - Proudfoot, M. - Cui, H. - Flick, R. - Anderson, W. F. - Joachimiak, A. - Galperin, M. Y. - Savchenko, A. - Yakunin, A. F.
Journal: J Mol Biol

Cyclic diguanylate (or bis-(3'-5') cyclic dimeric guanosine monophosphate; c-di-GMP) is a ubiquitous second messenger that regulates diverse cellular functions, including motility, biofilm formation, cell cycle progression, and virulence in bacteria. In the cell, degradation of c-di-GMP is catalyzed by highly specific EAL domain phosphodiesterases whose catalytic mechanism is still unclear. Here, we purified 13 EAL domain proteins from various organisms and demonstrated that their catalytic activity is associated with the presence of 10 conserved EAL domain residues. The crystal structure of the TBD1265 EAL domain was determined in free state (1.8 A) and in complex with c-di-GMP (2.35 A), and unveiled the role of conserved residues in substrate binding and catalysis. The structure revealed the presence of two metal ions directly coordinated by six conserved residues, two oxygens of c-di-GMP phosphate, and potential catalytic water molecule. Our results support a two-metal-ion catalytic mechanism of c-di-GMP hydrolysis by EAL domain phosphodiesterases.

post to: CiteULike

Chloroplast Import Signals: The Length Requirement for Translocation In Vitro and In Vivo.

Publication Date: 2010 Aug 3 PMID: 20688079
Authors: Bionda, T. - Tillmann, B. - Simm, S. - Beilstein, K. - Ruprecht, M. - Schleiff, E.
Journal: J Mol Biol

Protein translocation of cytosolically synthesized proteins requires signals for both targeting of precursor proteins to the surface of the respective compartment and their transfer across its membrane. In contrast to signals for peroxisomal and endoplasmic reticulum translocation, the signals for mitochondrial and chloroplast transport are less well defined with respect to length and amino acid requirements. To study the properties of signals required for translocation into chloroplasts in vitro and in vivo, we used fusion proteins composed of transit peptides and the Ig-like module of the muscle protein titin as passenger. We observed that about 60 amino acids-longer than the transit peptide length of many experimentally confirmed chloroplast proteins-are required for efficient translocation. However, within native chloroplast precursor proteins with transit peptides shorter than 60 amino acids, extension appears to be present as they are efficiently imported into organelles. In addition, the interaction of an unfolded polypeptide stretch of 60 or more amino acids with receptors at the chloroplast surface results in the unidirectionality of protein translocation into chloroplasts even in the presence of a competing C-terminal peroxisomal targeting signal. These findings prove the existing ideas that initial targeting is defined by the N-terminal signal and that the C-terminal signal is sensed only subsequently.

post to: CiteULike

Crystal structure of the 2-oxoglutarate- and Fe(II)-dependent lysyl hydroxylase JMJD6.

Publication Date: 2010 Aug 13 PMID: 20684070
Authors: Mantri, M. - Krojer, T. - Bagg, E. A. - Webby, C. J. - Butler, D. S. - Kochan, G. - Kavanagh, K. L. - Oppermann, U. - McDonough, M. A. - Schofield, C. J.
Journal: J Mol Biol

Lysyl and prolyl hydroxylations are well-known post-translational modifications to animal and plant proteins with extracellular roles. More recent work has indicated that the hydroxylation of intracellular animal proteins may be common. JMJD6 catalyses the iron- and 2-oxoglutarate-dependent hydroxylation of lysyl residues in arginine-serine-rich domains of RNA splicing-related proteins. We report crystallographic studies on the catalytic domain of JMJD6 in complex with Ni(II) substituting for Fe(II). Together with mutational studies, the structural data suggest how JMJD6 binds its lysyl residues such that it can catalyse C-5 hydroxylation rather than Nepsilon-demethylation, as for analogous enzymes.

MeSH Categories: Amino Acid Sequence, Amino Acid Substitution, Base Sequence, Catalytic Domain, Crystallography, X-Ray, DNA Primers/genetics, Humans, Iron/metabolism, Jumonji Domain-Containing Histone, Demethylases/*chemistry/genetics/metabolism, Ketoglutaric Acids/metabolism, Models, Molecular, Molecular Sequence Data, Mutagenesis, Site-Directed, Mutant Proteins/chemistry/genetics/metabolism, Nickel/metabolism, Procollagen-Lysine, 2-Oxoglutarate, 5-Dioxygenase/*chemistry/genetics/metabolism, Protein Folding, Recombinant Proteins/chemistry/genetics/metabolism, Sequence Homology, Amino Acid, Static Electricity

post to: CiteULike

Mutually-induced conformational switching of RNA and coat protein underpins efficient assembly of a viral capsid.

Publication Date: 2010 Aug 13 PMID: 20684044
Authors: Rolfsson, O. - Toropova, K. - Ranson, N. A. - Stockley, P. G.
Journal: J Mol Biol

Single-stranded RNA viruses package their genomes into capsids enclosing fixed volumes. We assayed the ability of bacteriophage MS2 coat protein to package large, defined fragments of its genomic, single-stranded RNA. We show that the efficiency of packaging into a T=3 capsid in vitro is inversely proportional to RNA length, implying that there is a free-energy barrier to be overcome during assembly. All the RNAs examined have greater solution persistence lengths than the internal diameter of the capsid into which they become packaged, suggesting that protein-mediated RNA compaction must occur during assembly. Binding ethidium bromide to one of these RNA fragments, which would be expected to reduce its flexibility, severely inhibited packaging, consistent with this idea. Cryo-EM structures of the capsids assembled in these experiments with the sub-genomic RNAs show a layer of RNA density beneath the coat protein shell but lack density for the inner RNA shell seen in the wild-type virion. The inner layer is restored when full-length virion RNA is used in the assembly reaction, implying that it becomes ordered only when the capsid is filled, presumably because of the effects of steric and/or electrostatic repulsions. The cryo-EM results explain the length dependence of packaging. In addition, they show that for the sub-genomic fragments the strongest ordered RNA density occurs below the coat protein dimers forming the icosahedral 5-fold axes of the capsid. There is little such density beneath the proteins at the 2-fold axes, consistent with our model in which coat protein dimers binding to RNA stem-loops located at sites throughout the genome leads to switching of their preferred conformations, thus regulating the placement of the quasi-conformers needed to build the T=3 capsid. The data are consistent with mutual chaperoning of both RNA and coat protein conformations, partially explaining the ability of such viruses to assemble so rapidly and accurately.

MeSH Categories: Base Sequence, Capsid/*chemistry/ultrastructure, Capsid Proteins/*chemistry/genetics, Cryoelectron Microscopy, DNA Primers/genetics, Imaging, Three-Dimensional, Levivirus/chemistry/enzymology/physiology/ultrastructure, Models, Molecular, Nucleic Acid Conformation, Protein Conformation, RNA, Viral/*chemistry/genetics, Recombinant Proteins/chemistry/genetics, Virus Assembly/*physiology

post to: CiteULike

Urate Is a Ligand for the Transcriptional Regulator PecS.

Publication Date: 2010 Aug 3 PMID: 20678501
Authors: Perera, I. C. - Grove, A.
Journal: J Mol Biol

PecS is a member of the MarR (multiple antibiotic resistance regulator) family, which has been shown in Erwinia to regulate the expression of virulence genes. MarR homologs typically bind a small molecule ligand, resulting in attenuated DNA binding. For PecS, the natural ligand has not been identified. We have previously shown that urate is a ligand for the Deinococcus radiodurans-encoded MarR homolog HucR (hypothetical uricase regulator) and identified residues responsible for ligand binding. We show here that all four residues involved in urate binding and propagation of conformational changes to DNA recognition helices are conserved in PecS homologs, suggesting that urate is the ligand for PecS. Consistent with this prediction, Agrobacterium tumefaciens PecS specifically binds urate, and urate attenuates DNA binding in vitro. PecS binds two operator sites in the intergenic region between the divergent pecS gene and pecM genes, one of which features two partially overlapping repeats to which PecS binds as a dimer on opposite faces of the duplex. Notably, urate dissociates PecS from cognate DNA, allowing transcription of both genes in vivo. Taken together, our data show that urate is a ligand for PecS and suggest that urate serves a novel function in signaling the colonization of a host plant.

post to: CiteULike

Structure and Engineering of l-Arabinitol 4-Dehydrogenase from Neurospora crassa.

Publication Date: 2010 Sep 10 PMID: 20655316
Authors: Bae, B. - Sullivan, R. P. - Zhao, H. - Nair, S. K.
Journal: J Mol Biol

l-Arabinitol 4-dehydrogenase (LAD) catalyzes the conversion of l-arabinitol into l-xylulose with concomitant NAD(+) reduction. It is an essential enzyme in the development of recombinant organisms that convert l-arabinose into fuels and chemicals using the fungal l-arabinose catabolic pathway. Here we report the crystal structure of LAD from the filamentous fungus Neurospora crassa at 2.6 A resolution. In addition, we created a number of site-directed variants of N. crassa LAD that are capable of utilizing NADP(+) as cofactor, yielding the first example of LAD with an almost completely switched cofactor specificity. This work represents the first structural data on any LAD and provides a molecular basis for understanding the existing literature on the substrate specificity and cofactor specificity of this enzyme. The engineered LAD mutants with altered cofactor specificity should be useful for applications in industrial biotechnology.

post to: CiteULike

HIV Fusion Peptide Penetrates, Disorders, and Softens T-Cell Membrane Mimics.

Publication Date: 2010 Sep 10 PMID: 20655315
Authors: Tristram-Nagle, S. - Chan, R. - Kooijman, E. - Uppamoochikkal, P. - Qiang, W. - Weliky, D. P. - Nagle, J. F.
Journal: J Mol Biol

This work investigates the interaction of N-terminal gp41 fusion peptide (FP) of human immunodeficiency virus type 1 (HIV-1) with model membranes in order to elucidate how FP leads to fusion of HIV and T-cell membranes. FP constructs were (i) wild-type FP23 (23 N-terminal amino acids of gp41), (ii) water-soluble monomeric FP that adds six lysines on the C-terminus of FP23 (FPwsm), and (iii) the C-terminus covalently linked trimeric version (FPtri) of FPwsm. Model membranes were (i) LM3 (a T-cell mimic), (ii) 1,2-dioleoyl-sn-glycero-3-phosphocholine, (iii) 1,2-dioleoyl-sn-glycero-3-phosphocholine/30 mol% cholesterol, (iv) 1,2-dierucoyl-sn-glycero-3-phosphocholine, and (v) 1,2-dierucoyl-sn-glycero-3-phosphocholine/30 mol% cholesterol. Diffuse synchrotron low-angle x-ray scattering from fully hydrated samples, supplemented by volumetric data, showed that FP23 and FPtri penetrate into the hydrocarbon region and cause membranes to thin. Depth of penetration appears to depend upon a complex combination of factors including bilayer thickness, presence of cholesterol, and electrostatics. X-ray data showed an increase in curvature in hexagonal phase 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, which further indicates that FP23 penetrates into the hydrocarbon region rather than residing in the interfacial headgroup region. Low-angle x-ray scattering data also yielded the bending modulus K(C), a measure of membrane stiffness, and wide-angle x-ray scattering yielded the S(xray) orientational order parameter. Both FP23 and FPtri decreased K(C) and S(xray) considerably, while the weak effect of FPwsm suggests that it did not partition strongly into LM3 model membranes. Our results are consistent with the HIV FP disordering and softening the T-cell membrane, thereby lowering the activation energy for viral membrane fusion.

post to: CiteULike

Crystal Structure of the GerBC Component of a Bacillus subtilis Spore Germinant Receptor.

Publication Date: 2010 Sep 10 PMID: 20654628
Authors: Li, Y. - Setlow, B. - Setlow, P. - Hao, B.
Journal: J Mol Biol

The nutrient germinant receptors (nGRs) of spores of Bacillus species are clusters of three proteins that play a critical role in triggering the germination of dormant spores in response to specific nutrient molecules. Here, we report the crystal structure of the C protein of the GerB germinant receptor, so-called GerBC, of Bacillus subtilis spores at 2.3 A resolution. The GerBC protein adopts a previously uncharacterized type of protein fold consisting of three distinct domains, each of which is centered by a beta sheet surrounded by multiple alpha helices. Secondary-structure prediction and structure-based sequence alignment suggest that the GerBC structure represents the prototype for C subunits of nGRs from spores of all Bacillales and Clostridiales species and defines two highly conserved structural regions in this family of proteins. GerBC forms an interlocked dimer in the crystalline state but is predominantly monomeric in solution, pointing to the possibility that GerBC oligomerizes as a result of either high local protein concentrations or interaction with other nGR proteins in spores. Our findings provide the first structural view of the nGR subunits and a molecular framework for understanding the architecture, conservation, and function of nGRs.

post to: CiteULike

Conformational Switching of the Diphtheria Toxin T Domain.

Publication Date: 2010 Sep 10 PMID: 20654627
Authors: Rodnin, M. V. - Kyrychenko, A. - Kienker, P. - Sharma, O. - Posokhov, Y. O. - Collier, R. J. - Finkelstein, A. - Ladokhin, A. S.
Journal: J Mol Biol

The diphtheria toxin T domain translocates the catalytic C domain across the endosomal membrane in response to acidification. To elucidate the role of histidine protonation in modulating pH-dependent membrane action of the T domain, we have used site-directed mutagenesis coupled with spectroscopic and physiological assays. Replacement of H257 with an arginine (but not with a glutamine) resulted in dramatic unfolding of the protein at neutral pH, accompanied by a substantial loss of helical structure and greatly increased exposure of the buried residues W206 and W281. This unfolding and spectral shift could be reversed by the interaction of the H257R mutant with model lipid membranes. Remarkably, this greatly unfolded mutant exhibited wild-type-like activity in channel formation, N-terminus translocation, and cytotoxicity assays. Moreover, membrane permeabilization caused by the H257R mutant occurs already at pH 6, where wild type protein is inactive. We conclude that protonation of H257 acts as a major component of the pH-dependent conformational switch, resulting in destabilization of the folded structure in solution and thereby promoting the initial membrane interactions necessary for translocation.

post to: CiteULike

Structure of the Complex between HER2 and an Antibody Paratope Formed by Side Chains from Tryptophan and Serine.

Publication Date: 2010 Sep 10 PMID: 20654626
Authors: Fisher, R. D. - Ultsch, M. - Lingel, A. - Schaefer, G. - Shao, L. - Birtalan, S. - Sidhu, S. S. - Eigenbrot, C.
Journal: J Mol Biol

Engineered antibody paratopes with limited sequence diversity permit assessment of the roles played by different amino acid side chains in creating the high-affinity, high-specificity interactions characteristic of antibodies. We describe a paratope raised against the human ErbB family member HER2, using a binary diversity tryptophan/serine library displayed on phage. Fab37 binds to the extracellular domain of HER2 with sub-nanomolar affinity. An X-ray structure at 3.2 A resolution reveals a contact paratope composed almost entirely of tryptophan and serine residues. Mutagenesis experiments reveal which of these side chains are more important for direct antigen interactions and which are more important for conformational flexibility. The crystal lattice contains an unprecedented trimeric arrangement of HER2 closely related to previously observed homodimers of the related epidermal growth factor receptor.

post to: CiteULike

Molecular Basis for Complement Recognition and Inhibition Determined by Crystallographic Studies of the Staphylococcal Complement Inhibitor (SCIN) Bound to C3c and C3b.

Publication Date: 2010 Sep 10 PMID: 20654625
Authors: Garcia, B. L. - Ramyar, K. X. - Tzekou, A. - Ricklin, D. - McWhorter, W. J. - Lambris, J. D. - Geisbrecht, B. V.
Journal: J Mol Biol

The human complement system plays an essential role in innate and adaptive immunity by marking and eliminating microbial intruders. Activation of complement on foreign surfaces results in proteolytic cleavage of complement component 3 (C3) into the potent opsonin C3b, which triggers a variety of immune responses and participates in a self-amplification loop mediated by a multi-protein assembly known as the C3 convertase. The human pathogen Staphylococcus aureus has evolved a sophisticated and potent complement evasion strategy, which is predicated upon an arsenal of potent inhibitory proteins. One of these, the staphylococcal complement inhibitor (SCIN), acts at the level of the C3 convertase (C3bBb) and impairs downstream complement function by trapping the convertase in a stable but inactive state. Previously, we have shown that SCIN binds C3b directly and competitively inhibits binding of human factor H and, to a lesser degree, that of factor B to C3b. Here, we report the co-crystal structures of SCIN bound to C3b and C3c at 7.5 and 3.5 A limiting resolution, respectively, and show that SCIN binds a critical functional area on C3b. Most significantly, the SCIN binding site sterically occludes the binding sites of both factor H and factor B. Our results give insight into SCIN binding to activated derivatives of C3, explain how SCIN can recognize C3b in the absence of other complement components, and provide a structural basis for the competitive C3b-binding properties of SCIN. In the future, this may suggest templates for the design of novel complement inhibitors based upon the SCIN structure.

post to: CiteULike

The M-Domain Controls Hsp104 Protein Remodeling Activity in an Hsp70/Hsp40-Dependent Manner.

Publication Date: 2010 Sep 10 PMID: 20654624
Authors: Sielaff, B. - Tsai, F. T.
Journal: J Mol Biol

Yeast Hsp104 is a ring-forming ATP-dependent protein disaggregase that, together with the cognate Hsp70 chaperone system, has the remarkable ability to rescue stress-damaged proteins from a previously aggregated state. Both upstream and downstream functions for the Hsp70 system have been reported, but it remains unclear how Hsp70/Hsp40 is coupled to Hsp104 protein remodeling activity. Hsp104 is a multidomain protein that possesses an N-terminal domain, an M-domain, and two tandem AAA(+) domains. The M-domain forms an 85-A long coiled coil and is a hallmark of the Hsp104 chaperone family. While the three-dimensional structure of Hsp104 has been determined, the function of the M-domain is unclear. Here, we demonstrate that the M-domain is essential for protein disaggregation, but dispensable for Hsp104 ATPase- and substrate-translocating activities. Remarkably, replacing the Hsp104 M-domain with that of bacterial ClpB, and vice versa, switches species specificity so that our chimeras now cooperate with the noncognate Hsp70/DnaK chaperone system. Our results demonstrate that the M-domain controls Hsp104 protein remodeling activities in an Hsp70/Hsp40-dependent manner, which is required to unleash Hsp104 protein disaggregating activity.

post to: CiteULike

Residue-Resolved Stability of Full-Consensus Ankyrin Repeat Proteins Probed by NMR.

Publication Date: 2010 Sep 10 PMID: 20654623
Authors: Wetzel, S. K. - Ewald, C. - Settanni, G. - Jurt, S. - Pluckthun, A. - Zerbe, O.
Journal: J Mol Biol

We investigated the stability determinants and the unfolding characteristics of full-consensus designed ankyrin repeat proteins (DARPins) by NMR. Despite the repeating sequence motifs, the resonances could be fully assigned using (2)H,(15)N,(13)C triple-labeled proteins. To remove further ambiguities, we attached paramagnetic spin labels to either end of these elongated proteins, which attenuate the resonances of the spatially closest residues. Deuterium exchange experiments of DARPins with two and three internal repeats between N- and C-terminal capping repeats (NI(2)C, NI(3)C) and NI(3)C_Mut5, where the C-cap had been reengineered, indicate that the stability of the full-consensus ankyrin repeat proteins is strongly dependent on the coupling between repeats, as the stabilized cap decreases the exchange rate throughout the whole protein. Some amide protons require more than a year to exchange at 37 degrees C, highlighting the extraordinary stability of the proteins. Denaturant-induced unfolding, followed by deuterium exchange, chemical shift change, and heteronuclear nuclear Overhauser effects, is consistent with an Ising-type description of equilibrium folding for NI(3)C_Mut5, while for native-state deuterium exchange, we postulate local fluctuations to dominate exchange as unfolding events are too slow in these very stable proteins. The location of extraordinarily slowly exchanging protons indicates a very stable core structure in the DARPins that combines hydrophobic shielding with favorable electrostatic interactions. These investigations help the understanding of repeat protein architecture and the further design of DARPins for biomedical applications where high stability is required.

post to: CiteULike

The Role of the T7 Gp2 Inhibitor of Host RNA Polymerase in Phage Development.

Publication Date: 2010 Sep 10 PMID: 20650282
Authors: Savalia, D. - Robins, W. - Nechaev, S. - Molineux, I. - Severinov, K.
Journal: J Mol Biol

Bacteriophage T7 relies on its own RNA polymerase (RNAp) to transcribe its middle and late genes. Early genes, which include the viral RNAp gene, are transcribed by the host RNAp from three closely spaced strong promoters-A1, A2, and A3. One middle T7 gene product, gp2, is a strong inhibitor of the host RNAp. Gp2 is essential and is required late in infection, during phage DNA packaging. Here, we explore the role of gp2 in controlling host RNAp transcription during T7 infection. We demonstrate that in the absence of gp2, early viral transcripts continue to accumulate throughout the infection. Decreasing transcription from early promoter A3 is sufficient to make gp2 dispensable for phage infection. Gp2 also becomes dispensable when an antiterminating element boxA, located downstream of early promoters, is deleted. The results thus suggest that antiterminated transcription by host RNAp from the A3 promoter is interfering with phage development and that the only essential role for gp2 is to prevent this transcription.

post to: CiteULike

Multiplexing RMCE: Versatile Extensions of the Flp-Recombinase-Mediated Cassette-Exchange Technology.

Publication Date: 2010 Sep 10 PMID: 20650281
Authors: Turan, S. - Kuehle, J. - Schambach, A. - Baum, C. - Bode, J.
Journal: J Mol Biol

There are strong indications, but as yet no proof, that extended 48-bp Flp recombinase targets (FRTs) represent unique targets in all eukaryotic genomes investigated, and that recombinase-mediated cassette exchange is not hampered by the occurrence of genomic pseudo sites. This encouraged the present study in which we explore the feasibility of exchanging, in a given cell, two distinct genomically anchored cassettes, each flanked by a unique set of two heterospecific FRT sites. Mutant FRTs have to meet two major prerequisites for successful recombinase-mediated cassette exchange: (i) a self-recognition capacity comparable to a pair of FRT wild-type sites (FRTxFRT), and (ii) a negligible cross-interaction if part of a set of heterospecific sites (F'xF). We apply a two-step strategy to explore various newly created FRT spacer mutants for these properties. As a result of our screening steps, we identify combinations of sites that are successfully applied to parallel Flp-mediated genomic targeting ("multiplexing") reactions (i.e., the simultaneous exchange of two separate target cassettes in a given cell).

post to: CiteULike

Cooperative Binding of MgATP and MgADP in the Trimeric P(II) Protein GlnK2 from Archaeoglobus fulgidus.

Publication Date: 2010 Sep 10 PMID: 20643148
Authors: Helfmann, S. - Lu, W. - Litz, C. - Andrade, S. L.
Journal: J Mol Biol

P(II)-like proteins, such as GlnK, found in a wide variety of organisms from prokaryotes to plants constitute a family of cytoplasmic signaling proteins that play a central regulatory role in the assimilation of nitrogen for biosyntheses. They specifically bind and are modulated by effector molecules such as adenosine triphosphate, adenosine diphosphate and 2-oxoglutarate. Their highly conserved, trimeric structure suggests that cooperativity in effector binding might be the basis for the ability to integrate and respond to a wide range of concentrations, but to date no direct quantification of this cooperative behavior has been presented. The hyperthermophilic archaeon Archaeoglobus fulgidus contains three GlnK proteins, functionally associated with ammonium transport proteins (Amt). We have characterized GlnK2 and its interaction with effectors by high-resolution X-ray crystallography and isothermal titration calorimetry. Binding of adenosine nucleotides resulted in distinct, cooperative behavior for ATP and ADP. While 2-oxoglutarate has been shown to interact with other GlnK proteins, GlnK2 was completely insensitive to this key indicator of a low level of intracellular nitrogen. These findings point to different regulation and modulation patterns and add to our understanding of the flexibility and versatility of the GlnK family of signaling proteins.

post to: CiteULike

Computational Mapping of Anchoring Spots on Protein Surfaces.

Publication Date: 2010 Sep 10 PMID: 20643147
Authors: Ben-Shimon, A. - Eisenstein, M.
Journal: J Mol Biol

Protein-protein and protein-peptide interactions are often controlled by few strong contacts that involve hot spot residues. Computational detection of such contacts, termed here anchoring spots, is important for understanding recognition processes and for predicting interactions; it is an essential step in designing interaction interfaces and therapeutic agents. We describe ANCHORSMAP, an algorithm for computational mapping of amino acid side chains on protein surfaces. The algorithm consists of two stages: A geometry based stage (LSMdet), in which sub-pockets adequate for binding single side chains are detected and amino acid probes are scattered near them, and an energy based stage in which optimal positions of the probes are determined through repeated energy minimization and clustering of nearby poses and their DeltaG are calculated. ANCHORSMAP employs a new function for DeltaG calculations, which is specifically designed for the context of protein-protein recognition by introducing a correction in the electrostatic energy term that compensates for the dielectric shielding exerted by a hypothetical protein bound to the probe. The algorithm successfully detects known anchoring sites and accurately positions the probes. The calculated DeltaG rank high the correct anchoring spots in maps produced for unbound proteins. We find that Arg, Trp, Glu and Tyr, which are favorite hot spot residues, are also more selective of their binding environment. The usefulness of anchoring spots mapping is demonstrated by detecting the binding surfaces in the protein-protein complex barnase/barstar and the protein-peptide complex kinase/PKI, and by identifying phenylalanine anchoring sites on the surface of the nuclear transporter NTF2, C-terminus anchors on PDZ domains and phenol anchors on thermolysin. Finally, we discuss the role of anchoring spots in molecular recognition processes.

post to: CiteULike

The PAX3 Paired Domain and Homeodomain Function as a Single Binding Module In Vivo to Regulate Subnuclear Localization and Mobility by a Mechanism That Requires Base-Specific Recognition.

Publication Date: 2010 Sep 10 PMID: 20643146
Authors: Corry, G. N. - Raghuram, N. - Missiaen, K. K. - Hu, N. - Hendzel, M. J. - Underhill, D. A.
Journal: J Mol Biol

The transcription factor PAX3 is essential for myogenesis and neural crest development, and is one of several genes mutated in human Waardenburg syndrome. Analysis of disease-causing missense mutations in PAX3 has established the interdependence of its two DNA-binding domains, the paired domain (PD) and the homeodomain (HD), as well as defects in localization and mobility. Paradoxically, mutants that retained DNA binding activity exhibited the greatest defects in localization and mobility, regardless of the domain in which they reside. In the present study, structure-function analyses were used to determine the mechanistic basis of this effect. In the context of the isolated DNA-binding domains, HD mutants adopted an increase in mobility proportional to their loss in DNA binding, while PD mutants continued to display the inverse relationship observed in the full-length protein. At the structural level, this reflected an unexpected dependence on base-specific contacts in the PD, whereas HD mobility was more severely affected by loss of backbone contacts, as has been observed with other DNA-binding proteins. This requires that the HD switch to a base-specific mode in the full-length protein. Moreover, both domains underwent substantial reduction in mobility and altered localization when in a contiguous polypeptide with the endogenous linker segment. Notably, although the HD conferred localization to heterochromatin, this activity was masked when linked to the PD, despite the absence of determinants for subnuclear compartmentalization in the PD or linker. Last, the propensity for PAX3 heterochromatin localization was modulated by sequences at the amino and carboxy termini, supporting a model in which alternate conformations lead to unmasking of the HD. These data indicate that the PD and the HD functionally interact in vivo and behave as a single binding module whose mobility and localization are dependent on sequence-specific contacts.

post to: CiteULike

Orientation of Small Multidrug Resistance Transporter Subunits in the Membrane: Correlation with the Positive-Inside Rule.

Publication Date: 2010 Sep 10 PMID: 20643145
Authors: Kolbusz, M. A. - Ter Horst, R. - Slotboom, D. J. - Lolkema, J. S.
Journal: J Mol Biol

Small multidrug resistance (SMR) transport proteins provide a model for the evolution of larger two-domain transport proteins. The orientation in the membrane of 27 proteins from the SMR family was determined using the reporter fusion technique. Nine members were encoded monocistronically (singles) and shown to insert in both orientations (dual topology). Eighteen members were encoded in pairs on the chromosome and shown to insert in fixed orientations; the two proteins in each pair invariably had opposite orientations in the membrane. Interaction between the two proteins in pairs was demonstrated by copurification. The orientation in the membrane of either protein in the pair was affected only marginally by the presence of the other protein. For the proteins in pairs, the orientation in the membrane correlated well with the distribution of positively charges residues (R+K) over the cytoplasmic and extracellular loops (positive-inside rule). In contrast, dual-topology insertion of the singles was predicted less well by the positive-inside rule. Three singles were predicted to insert in a single orientation with the N-terminus and the C-terminus at the extracellular side of the membrane. Analysis of charge distributions suggests the requirement of a threshold number of charges in the cytoplasmic loops for the positive-inside rule to be of predictive value. It is concluded that a combined analysis of gene organization on the chromosome and phylogeny is sufficient to distinguish between fixed or dual topology of SMR members and, probably, similar types of membrane proteins. The positive-inside rule can be used to predict the orientation of members in pairs, but is not suitable as a sole predictor of dual topology.

post to: CiteULike

Relative Affinity of Calcium Pump Isoforms for Phospholamban Quantified by Fluorescence Resonance Energy Transfer.

Publication Date: 2010 Sep 10 PMID: 20643144
Authors: Hou, Z. - Robia, S. L.
Journal: J Mol Biol

To investigate the regulation of SERCA1a [sarco(endo)plasmic reticulum calcium ATPase] and SERCA2a calcium pump isoforms by phospholamban (PLB), we quantified PLB-SERCA interactions by fluorescence resonance energy transfer (FRET) in live cells. For both SERCA1a and SERCA2a, FRET to PLB increased with increasing protein expression level to a maximum value corresponding to a probe separation distance of 64 A. The data indicate that the respective regulatory complexes assume the same overall quaternary conformation. However, FRET measurements also revealed that PLB has a 50% higher apparent affinity for SERCA1a relative to SERCA2a. The results suggest that despite the structural similarities of the respective regulatory complexes, there is preferential binding of PLB to SERCA1a over SERCA2a. This apparent selectivity may have implications for biochemical studies in which SERCA1a is used as a substitute for SERCA2a. It may also be an important strategic consideration for therapeutic overexpression of SERCA isoforms in cardiac muscle.

post to: CiteULike

Structural Evidence that Peroxiredoxin Catalytic Power Is Based on Transition-State Stabilization.

Publication Date: 2010 Sep 10 PMID: 20643143
Authors: Hall, A. - Parsonage, D. - Poole, L. B. - Karplus, P. A.
Journal: J Mol Biol

Peroxiredoxins (Prxs) are important peroxidases associated with both antioxidant protection and redox signaling. They use a conserved Cys residue to reduce peroxide substrates. The Prxs have a remarkably high catalytic efficiency that makes them a dominant player in cell-wide peroxide reduction, but the origins of their high activity have been mysterious. We present here a novel structure of human PrxV at 1.45 A resolution that has a dithiothreitol bound in the active site with its diol moiety mimicking the two oxygens of a peroxide substrate. This suggests diols and similar di-oxygen compounds as a novel class of competitive inhibitors for the Prxs. Common features of this and other structures containing peroxide, peroxide-mimicking ligands, or peroxide-mimicking water molecules reveal hydrogen bonding and steric factors that promote its high reactivity by creating an oxygen track along which the peroxide oxygens move as the reaction proceeds. Key insights include how the active-site microenvironment activates both the peroxidatic cysteine side chain and the peroxide substrate and how it is exquisitely well suited to stabilize the transition state of the in-line S(N)2 substitution reaction that is peroxidation.

post to: CiteULike

Methylated DNA Causes a Physical Block to Replication Forks Independently of Damage Signalling, O(6)-Methylguanine or DNA Single-Strand Breaks and Results in DNA Damage.

Publication Date: 2010 Sep 10 PMID: 20643142
Authors: Groth, P. - Auslander, S. - Majumder, M. M. - Schultz, N. - Johansson, F. - Petermann, E. - Helleday, T.
Journal: J Mol Biol

Even though DNA alkylating agents have been used for many decades in the treatment of cancer, it remains unclear what happens when replication forks encounter alkylated DNA. Here, we used the DNA fibre assay to study the impact of alkylating agents on replication fork progression. We found that the alkylator methyl methanesulfonate (MMS) inhibits replication elongation in a manner that is dose dependent and related to the overall alkylation grade. Replication forks seem to be completely blocked as no nucleotide incorporation can be detected following 1 h of MMS treatment. A high dose of 5 mM caffeine, inhibiting most DNA damage signalling, decreases replication rates overall but does not reverse MMS-induced replication inhibition, showing that the replication block is independent of DNA damage signalling. Furthermore, the block of replication fork progression does not correlate with the level of DNA single-strand breaks. Overexpression of O(6)-methylguanine (O6meG)-DNA methyltransferase protein, responsible for removing the most toxic alkylation, O6meG, did not affect replication elongation following exposure to N-methyl-N'-nitro-N-nitrosoguanidine. This demonstrates that O6meG lesions are efficiently bypassed in mammalian cells. In addition, we find that MMS-induced gammaH2AX foci co-localise with 53BP1 foci and newly replicated areas, suggesting that DNA double-strand breaks are formed at MMS-blocked replication forks. Altogether, our data suggest that N-alkylations formed during exposure to alkylating agents physically block replication fork elongation in mammalian cells, causing formation of replication-associated DNA lesions, likely double-strand breaks.

post to: CiteULike

Use of RNA Tertiary Interaction Modules for the Crystallisation of the Spliceosomal snRNP Core Domain.

Publication Date: 2010 Sep 10 PMID: 20643141
Authors: Leung, A. K. - Kambach, C. - Kondo, Y. - Kampmann, M. - Jinek, M. - Nagai, K.
Journal: J Mol Biol

RNA is known to perform diverse roles in the cell, often as ribonucleoprotein (RNP) particles. While the crystal structure of these RNP particles could provide crucial insights into their functions, crystallographic work on RNP complexes is often hampered by difficulties in obtaining well-diffracting crystals. The small nuclear ribonucleoprotein (snRNP) core domain, acting as an assembly nucleus for the maturation of snRNPs, plays a crucial role in the biogenesis of four of the spliceosomal snRNPs. We have succeeded in crystallising the human U4 snRNP core domain containing seven Sm proteins and a truncated U4 snRNA variant. The most critical factor in our success in the crystallisation was the introduction of various tertiary interaction modules into the RNA that could promote crystal packing without altering the core structure. Here, we describe various strategies employed in our crystallisation effort that could be applied to crystallisation of other RNP particles.

post to: CiteULike

Translation Driven by Picornavirus IRES Is Hampered from Sindbis Virus Replicons: Rescue by Poliovirus 2A Protease.

Publication Date: 2010 Sep 10 PMID: 20643140
Authors: Sanz, M. A. - Welnowska, E. - Redondo, N. - Carrasco, L.
Journal: J Mol Biol

Alphavirus replicons are very useful for analyzing different aspects of viral molecular biology. They are also useful tools in the development of new vaccines and highly efficient expression of heterologous genes. We have investigated the translatability of Sindbis virus (SV) subgenomic mRNA bearing different 5'-untranslated regions, including several viral internal ribosome entry sites (IRESs) from picornaviruses, hepatitis C virus, and cricket paralysis virus. Our findings indicate that all these IRES-containing mRNAs are initially translated in culture cells transfected with the corresponding SV replicon but their translation is inhibited in the late phase of SV replication. Notably, co-expression of different poliovirus (PV) non-structural genes reveals that the protease 2A (2A(pro)) is able to increase translation of subgenomic mRNAs containing the PV or encephalomyocarditis virus IRESs but not of those of hepatitis C virus or cricket paralysis virus. A PV 2A(pro) variant deficient in eukaryotic initiation factor (eIF) 4GI cleavage or PV protease 3C, neither of which cleaves eIF4GI, does not increase picornavirus IRES-driven translation, whereas L protease from foot-and-mouth disease virus also rescues translation. These findings suggest that the replicative foci of SV-infected cells where translation takes place are deficient in components necessary to translate IRES-containing mRNAs. In the case of picornavirus IRESs, cleavage of eIF4GI accomplished by PV 2A(pro) or foot-and-mouth disease virus protease L rescues this inhibition. eIF4GI co-localizes with ribosomes both in cells electroporated with SV replicons bearing the picornavirus IRES and in cells co-electroporated with replicons that express PV 2A(pro). These findings support the idea that eIF4GI cleavage is necessary to rescue the translation driven by picornavirus IRESs in baby hamster kidney cells that express SV replicons.

post to: CiteULike

Long-Distance Communication in the HDV Ribozyme: Insights from Molecular Dynamics and Experiments.

Publication Date: 2010 Sep 10 PMID: 20643139
Authors: Veeraraghavan, N. - Bevilacqua, P. C. - Hammes-Schiffer, S.
Journal: J Mol Biol

The hepatitis delta virus ribozyme is a small, self-cleaving RNA with a compact tertiary structure and buried active site that is important in the life cycle of the virus. The ribozyme's function in nature is to cleave an internal phosphodiester bond and linearize concatemers during rolling circle replication. Crystal structures of the ribozyme have been solved in both pre-cleaved and post-cleaved (product) forms and reveal an intricate network of interactions that conspire to catalyze bond cleavage. In addition, extensive biochemical studies have been performed to work out a mechanism for bond cleavage in which C75 and a magnesium ion catalyze the reaction by general acid-base chemistry. One issue that has remained unclear in this ribozyme and in other ribozymes is the nature of long-distance communication between peripheral regions of the RNA and the buried active site. We performed molecular dynamics simulations on the hepatitis delta virus ribozyme in the product form and assessed communication between a distal structural portion of the ribozyme-the protonated C41 base triple-and the active site containing the critical C75. We varied the ionization state of C41 in both the wild type and a C41 double mutant variant and determined the impact on the active site. In all four cases, effects at the active site observed in the simulations agree with experimental studies on ribozyme activity. Overall, these studies indicate that small functional RNAs have the potential to communicate interactions over long distances and that wild-type RNAs may have evolved ways to prevent such interactions from interfering with catalysis.

post to: CiteULike

Nebulin: A Study of Protein Repeat Evolution.

Publication Date: 2010 Sep 10 PMID: 20643138
Authors: Bjorklund, A. K. - Light, S. - Sagit, R. - Elofsson, A.
Journal: J Mol Biol

Protein domain repeats are common in proteins that are central to the organization of a cell, in particular in eukaryotes. They are known to evolve through internal tandem duplications. However, the understanding of the underlying mechanisms is incomplete. To shed light on repeat expansion mechanisms, we have studied the evolution of the muscle protein Nebulin, a protein that contains a large number of actin-binding nebulin domains. Nebulin proteins have evolved from an invertebrate precursor containing two nebulin domains. Repeat regions have expanded through duplications of single domains, as well as duplications of a super repeat (SR) consisting of seven nebulins. We show that the SR has evolved independently into large regions in at least three instances: twice in the invertebrate Branchiostoma floridae and once in vertebrates. In-depth analysis reveals several recent tandem duplications in the Nebulin gene. The events involve both single-domain and multidomain SR units or several SR units. There are single events, but frequently the same unit is duplicated multiple times. For instance, an ancestor of human and chimpanzee underwent two tandem duplications. The duplication junction coincides with an Alu transposon, thus suggesting duplication through Alu-mediated homologous recombination. Duplications in the SR region consistently involve multiples of seven domains. However, the exact unit that is duplicated varies both between species and within species. Thus, multiple tandem duplications of the same motif did not create the large Nebulin protein. Finally, analysis of segmental duplications in the human genome reveals that duplications are more common in genes containing domain repeats than in those coding for nonrepeated proteins. In fact, segmental duplications are found three to six times more often in long repeated genes than expected by chance.

post to: CiteULike

Molecular Basis for the Recognition of Phosphorylated STAT1 by Importin alpha5.

Publication Date: 2010 Sep 10 PMID: 20643137
Authors: Nardozzi, J. - Wenta, N. - Yasuhara, N. - Vinkemeier, U. - Cingolani, G.
Journal: J Mol Biol

Interferon-gamma stimulation triggers tyrosine phosphorylation of the transcription factor STAT1 at position 701, which is associated with switching from carrier-independent nucleocytoplasmic shuttling to carrier-mediated nuclear import. Unlike most substrates that carry a classical nuclear localization signal (NLS) and bind to importin alpha1, STAT1 possesses a nonclassical NLS recognized by the isoform importin alpha5. In the present study, we have analyzed the mechanisms by which importin alpha5 binds phosphorylated STAT1 (pSTAT1). We found that a homodimer of pSTAT1 is recognized by one equivalent of importin alpha5 with K(d)=191+/-20 nM. Whereas tyrosine phosphorylation at position 701 is essential to assemble a pSTAT1-importin alpha5 complex, the phosphate moiety is not a direct binding determinant for importin alpha5. In contrast to classical NLS substrates, pSTAT1 binding to importin alpha5 is not displaced by the N-terminal importin beta binding domain and requires the importin alpha5 C-terminal acidic tail (505-EEDD-508). A local unfolding of importin alpha5 Armadillo (ARM) repeat 10 accompanies high-affinity binding to pSTAT1. This unfolding is mediated by a single conserved tyrosine at position 476 of importin alpha5, which is inserted between ARM repeat 10 helices H1-H2-H3, thereby preventing intramolecular helical stacking essential to stabilize the folding conformation of ARM 10. Introducing a glycine at this position, as in importin alpha1, disrupts high-affinity binding to pSTAT1, suggesting that pSTAT1 recognition is dependent on the intrinsic flexibility of ARM 10. Using the quantitative stoichiometry and binding data presented in this article, together with mutational information available in the literature, we propose that importin alpha5 binds between two STAT1 monomers, with two major binding determinants in the SH2 and DNA binding domains. In vitro, this model is supported by the observation that a 38-mer DNA oligonucleotide containing two tandem cfosM67 promoters can displace importin alpha5 from pSTAT1, suggesting a possible role for DNA in releasing activated STAT1 in the cell nucleus.

post to: CiteULike

Allosteric remodelling of the histone H3 binding pocket in the Pygo2 PHD finger triggered by its binding to the B9L/BCL9 co-factor.

Publication Date: 2010 Sep 3 PMID: 20637214
Authors: Miller, T. C. - Rutherford, T. J. - Johnson, C. M. - Fiedler, M. - Bienz, M.
Journal: J Mol Biol

The Zn-coordinated PHD fingers of Pygopus (Pygo) proteins are critical for beta-catenin-dependent transcriptional switches in normal and malignant tissues. They bind to methylated histone H3 tails, assisted by their BCL9 co-factors whose homology domain 1 (HD1) binds to the rear PHD surface. Although histone-binding residues are identical between the two human Pygo paralogs, we show here that Pygo2 complexes exhibit slightly higher binding affinities for methylated histone H3 tail peptides than Pygo1 complexes. We solved the crystal structure of the Pygo2 PHD-BCL9-2 HD1 complex, which revealed paralog-specific interactions in its PHD-HD1 interface that could contribute indirectly to its elevated affinity for the methylated histone H3 tail. Interestingly, using NMR spectroscopy, we discovered that HD1 binding to PHD triggers an allosteric communication with a conserved isoleucine residue that lines the binding channel for histone H3 threonine 3 (T3), the link between the two adjacent binding pockets accommodating histone H3 alanine 1 and methylated lysine 4, respectively. This modulates the surface of the T3 channel, providing a plausible explanation as to how BCL9 co-factors binding to Pygo PHD fingers impact indirectly on their histone binding affinity. Intriguingly, this allosteric modulation of the T3 channel is propagated through the PHD structural core by a highly conserved tryptophan, the signature residue defining the PHD subclass of Zn fingers, which suggests that other PHD proteins may also be assisted by co-factors in their decoding of modified histone H3 tails.

post to: CiteULike

A mechanism of release of calreticulin from cells during apoptosis.

Publication Date: 2010 Sep 3 PMID: 20624402
Authors: Tarr, J. M. - Young, P. J. - Morse, R. - Shaw, D. J. - Haigh, R. - Petrov, P. G. - Johnson, S. J. - Winyard, P. G. - Eggleton, P.
Journal: J Mol Biol

Calreticulin (CRT) is an endoplasmic reticulum (ER) chaperone responsible for glycoprotein folding and Ca(2+) homeostasis. CRT also has extracellular functions, e.g. tumor and apoptotic cell recognition and wound healing, but the mechanism of CRT extracellular release is unknown. Cytosolic localization of CRT is determined by signal peptide and subsequent retrotranslocation of CRT into the cytoplasm. Here, we show that under apoptotic stress conditions, the cytosolic concentration of CRT increases and associates with phosphatidylserine (PS) in a Ca(2)(+)-dependent manner. PS distribution is regulated by aminophospholipid translocase (APLT), which maintains PS on the cytosolic side of the cell membrane. APLT is sensitive to redox modifications of its SH groups by reactive nitrogen species. During apoptosis, both CRT expression and the concentration of nitric oxide (NO) increase. By using S-nitroso-l-cysteine-ethyl-ester, an intracellular NO donor and inhibitor of APLT, we showed that PS and CRT externalization occurred together in an S-nitrosothiol-dependent and caspase-independent manner. Furthermore, the CRT and PS are relocated as punctate clusters on the cell surface. Thus, CRT induced nitrosylation and its externalization with PS could explain how CRT acts as a bridging molecule during apoptotic cell clearance.

post to: CiteULike

Interaction of the Hsp110 molecular chaperones from S. cerevisiae with substrate protein.

Publication Date: 2010 Sep 3 PMID: 20624400
Authors: Polier, S. - Hartl, F. U. - Bracher, A.
Journal: J Mol Biol

Hsp110 proteins act as nucleotide exchange factors of the molecular chaperone Hsp70 in eukaryotes. In addition, they have been reported to stabilize unfolded proteins for subsequent refolding. Hsp110 proteins belong to the Hsp70 superfamily and, in analogy to Hsp70, the substrate-binding site was proposed to be located at the interface of the beta-sandwich domain and the three-helix-bundle domain. Saccharomyces cerevisiae has two closely related cytosolic isoforms of Hsp110, Sse1p and Sse2p. Under normal growth conditions, Sse1p is the predominant form. Sse2p is induced under stress conditions, such as heat shock. Consistent with these findings, we find that Sse2p has increased temperature stability. Both Sse1p and Sse2p accelerate nucleotide exchange on the yeast Hsp70 Ssa1p. Furthermore, Sse1p and Sse2p effectively compete for binding of unfolded luciferase. In contrast to Sse1p, however, Sse2p fails to stabilize this model substrate under thermal stress for subsequent Hsp70-mediated refolding. Using a domain shuffling approach, we show that both the nucleotide-binding domain and the beta-sandwich domain of Sse1p are required to preserve nonnative luciferase in a folding-competent state. Our findings suggest that Sse1p must undergo partial unfolding for efficient protection of luciferase, and that the beta-sandwich domain of Sse1p acts as an intramolecular chaperone for refolding of the nucleotide-binding domain. Under extreme stress conditions, Sse2p appears to take over the nucleotide exchange factor function of Sse1p and might promote the controlled aggregation of stress-denatured proteins.

post to: CiteULike

Ion selectivity of the KcsA channel: a perspective from multi-ion free energy landscapes.

Publication Date: 2010 Sep 3 PMID: 20624398
Authors: Egwolf, B. - Roux, B.
Journal: J Mol Biol

Potassium (K(+)) channels are specialized membrane proteins that are able to facilitate and regulate the conduction of K(+) through cell membranes. Comprising five specific cation binding sites (S(0)-S(4)) formed by the backbone carbonyl groups of conserved residues common to all K(+) channels, the narrow selectivity filter allows fast conduction of K(+) while being highly selective for K(+) over Na(+). To extend our knowledge of the microscopic mechanism underlying selectivity in K(+) channels, we characterize the free energy landscapes governing the entry and translocation of a Na(+) or a K(+) from the extracellular side into the selectivity filter of KcsA. The entry process of an extracellular ion is examined in the presence of two additional K(+) in the pore, and the three-ion potential of mean force is computed using extensive all-atom umbrella sampling molecular dynamics simulations. A comparison of the potentials of mean force yields a number of important results. First, the free energy minima corresponding to configurations with extracellular K(+) or Na(+) in binding site S(0) or S(1) are similar in depth, suggesting that the thermodynamic selectivity governed by the free energy minima for those two binding sites is insignificant. Second, the free energy barriers between stable multi-ion configurations are generally higher for Na(+) than for K(+), implying that the kinetics of ion conduction is slower when a Na(+) enters the pore. Third, the region corresponding to binding site S(2) near the center of the narrow pore emerges as the most selective for K(+) over Na(+). In particular, while there is a stable minimum for K(+) in site S(2), Na(+) faces a steep free energy increase with no local free energy well in this region. Lastly, analysis shows that selectivity is not correlated with the overall coordination number of the ion entering the pore, but is predominantly affected by changes in the type of coordinating ligands (carbonyls versus water molecules). These results further highlight the importance of the central region near binding site S(2) in the selectivity filter of K(+) channels.

post to: CiteULike

Partially folded aggregation intermediates of human gammaD-, gammaC-, and gammaS-crystallin are recognized and bound by human alphaB-crystallin chaperone.

Publication Date: 2010 Aug 6 PMID: 20621668
Authors: Acosta-Sampson, L. - King, J.
Journal: J Mol Biol

Human gamma-crystallins are long-lived, unusually stable proteins of the eye lens exhibiting duplicated, double Greek key domains. The lens also contains high concentrations of the small heat shock chaperone alpha-crystallin, which suppresses aggregation of model substrates in vitro. Mature-onset cataract is believed to represent an aggregated state of partially unfolded and covalently damaged crystallins. Nonetheless, the lack of cell or tissue culture for anucleate lens fibers and the insoluble state of cataract proteins have made it difficult to identify the conformation of the human gamma-crystallin substrate species recognized by human alpha-crystallin. The three major human lens monomeric gamma-crystallins, gammaD, gammaC, and gammaS, all refold in vitro in the absence of chaperones, on dilution from denaturant into buffer. However, off-pathway aggregation of the partially folded intermediates competes with productive refolding. Incubation with human alphaB-crystallin chaperone during refolding suppressed the aggregation pathways of the three human gamma-crystallin proteins. The chaperone did not dissociate or refold the aggregated chains under these conditions. The alphaB-crystallin oligomers formed long-lived stable complexes with their gammaD-crystallin substrates. Using alpha-crystallin chaperone variants lacking tryptophans, we obtained fluorescence spectra of the chaperone-substrate complex. Binding of substrate gamma-crystallins with two or three of the four buried tryptophans replaced by phenylalanines showed that the bound substrate remained in a partially folded state with neither domain native-like. These in vitro results provide support for protein unfolding/protein aggregation models for cataract, with alpha-crystallin suppressing aggregation of damaged or unfolded proteins through early adulthood but becoming saturated with advancing age.

MeSH Categories: Binding Sites, Chemical Precipitation, Circular Dichroism, Humans, Models, Biological, Models, Molecular, Molecular Chaperones/chemistry/metabolism, Protein Binding/physiology, Protein Conformation, *Protein Folding, Recombinant Proteins/chemistry/isolation & purification/metabolism, Spectrometry, Fluorescence, alpha-Crystallin B Chain/chemistry/isolation & purification/*metabolism, gamma-Crystallins/*chemistry/isolation & purification/*metabolism

post to: CiteULike

Crystal structure of the dog lipocalin allergen Can f 2: implications for cross-reactivity to the cat allergen Fel d 4.

Publication Date: 2010 Aug 6 PMID: 20621650
Authors: Madhurantakam, C. - Nilsson, O. B. - Uchtenhagen, H. - Konradsen, J. - Saarne, T. - Hogbom, E. - Sandalova, T. - Gronlund, H. - Achour, A.
Journal: J Mol Biol

The dog lipocalin allergen Can f 2 is an important cause of allergic sensitization in humans worldwide. Here, the first crystal structure of recombinant rCan f 2 at 1.45 A resolution displays a classical lipocalin fold with a conserved Gly-Xaa-Trp motif, in which Trp19 stabilizes the overall topology of the monomeric rCan f 2. Phe38 and Tyr84 localized on the L1 and L5 loops, respectively, control access to the highly hydrophobic calyx. Although the rCan f 2 calyx is nearly identical with the aero-allergens MUP1, Equ c 1 and A2U from mouse, horse and rat, respectively, no IgE cross-reactivity was found using sera from five mono-sensitized subjects. However, clear IgE cross-reactivity was demonstrated between Can f 2 and the cat allergen Fel d 4, although they share less than 22% sequence identity. This suggests a role for these allergens in co-sensitization between cat- and dog-allergic patients.

MeSH Categories: Allergens/*chemistry/*immunology, Amino Acid Sequence, Animals, Antibody Specificity, Cats, Cross Reactions, Crystallization, Crystallography, X-Ray, Dogs, Female, Glycoproteins/*chemistry/*immunology, Humans, Lipocalins/*chemistry/immunology, Mice, Mice, Inbred BALB C, Models, Molecular, Molecular Sequence Data, Protein Conformation, Sequence Homology, Amino Acid

post to: CiteULike

The impact of viral RNA on assembly pathway selection.

Publication Date: 2010 Aug 13 PMID: 20621589
Authors: Morton, V. L. - Dykeman, E. C. - Stonehouse, N. J. - Ashcroft, A. E. - Twarock, R. - Stockley, P. G.
Journal: J Mol Biol

Many single-stranded RNA viruses self-assemble their protein containers around their genomes. The roles that the RNA plays in this assembly process have mostly been ignored, resulting in a protein-centric view of assembly that is unable to explain adequately the fidelity and speed of assembly in such viruses. Using bacteriophage MS2, we demonstrate here via a combination of mass spectrometry and kinetic modelling how viral RNA can bias assembly towards only a small number of the many possible assembly pathways, thus increasing assembly efficiency. Assembly reactions have been studied in vitro using phage coat protein dimers, the known building block of the T=3 shell, and short RNA stem-loops based on the translational operator of the replicase cistron, a 19 nt fragment (TR). Mass spectrometry has unambiguously identified two on-pathway intermediates in such reactions that have stoichiometry consistent with formation of either a particle 3-fold or 5-fold axis. These imply that there are at least two sub-pathways to the final capsid. The flux through each pathway is controlled by the length of the RNA stem-loop triggering the assembly reaction and this effect can be understood in structural terms. The kinetics of intermediate formation have been studied and show steady-state concentrations for intermediates between starting materials and the T=3 shell, consistent with an assembly process in which all the steps are in equilibrium. These data have been used to derive a kinetic model of the assembly reaction that in turn allows us to determine the dominant assembly pathways explicitly, and to estimate the effect of the RNA on the free energy of association between the assembling protein subunits. The results reveal that there are only a small number of dominant assembly pathways, which vary depending on the relative ratios of RNA and protein. These results suggest that the genomic RNA plays significant roles in defining the precise assembly sub-pathway followed to create the final capsid.

MeSH Categories: Base Sequence, Capsid/chemistry, Capsid Proteins/chemistry, Kinetics, Levivirus/chemistry/genetics/physiology, Macromolecular Substances/chemistry, Mass Spectrometry, Models, Molecular, Nucleic Acid Conformation, Protein Subunits, RNA, Viral/*chemistry/genetics, Thermodynamics, Virus Assembly/genetics/*physiology

post to: CiteULike

Assembly and maturation of the bacteriophage lambda procapsid: gpC is the viral protease.

Publication Date: 2010 Sep 3 PMID: 20620152
Authors: Medina, E. - Wieczorek, D. - Medina, E. M. - Yang, Q. - Feiss, M. - Catalano, C. E.
Journal: J Mol Biol

Viral capsids are robust structures designed to protect the genome from environmental insults and deliver it to the host cell. The developmental pathway for complex double-stranded DNA viruses is generally conserved in the prokaryotic and eukaryotic groups and includes a genome packaging step where viral DNA is inserted into a pre-formed procapsid shell. The procapsids self-assemble from monomeric precursors to afford a mature icosahedron that contains a single "portal" structure at a unique vertex; the portal serves as the hole through which DNA enters the procapsid during particle assembly and exits during infection. Bacteriophage lambda has served as an ideal model system to study the development of the large double-stranded DNA viruses. Within this context, the lambda procapsid assembly pathway has been reported to be uniquely complex involving protein cross-linking and proteolytic maturation events. In this work, we identify and characterize the protease responsible for lambda procapsid maturation and present a structural model for a procapsid-bound protease dimer. The procapsid protease possesses autoproteolytic activity, it is required for degradation of the internal "scaffold" protein required for procapsid self-assembly, and it is responsible for proteolysis of the portal complex. Our data demonstrate that these proteolytic maturation events are not required for procapsid assembly or for DNA packaging into the structure, but that proteolysis is essential to late steps in particle assembly and/or in subsequent infection of a host cell. The data suggest that the lambda-like proteases and the herpesvirus-like proteases define two distinct viral protease folds that exhibit little sequence or structural homology but that provide identical functions in virus development. The data further indicate that procapsid assembly and maturation are strongly conserved in the prokaryotic and eukaryotic virus groups.

post to: CiteULike

Crystal structure of glyceraldehyde-3-phosphate dehydrogenase 1 from methicillin-resistant Staphylococcus aureus MRSA252 provides novel insights into substrate binding and catalytic mechanism.

Publication Date: 2010 Sep 3 PMID: 20620151
Authors: Mukherjee, S. - Dutta, D. - Saha, B. - Das, A. K.
Journal: J Mol Biol

The dreaded pathogen Staphylococcus aureus is one of the causes of morbidity and mortality worldwide. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), one of the key glycolytic enzymes, is irreversibly oxidized under oxidative stress and is responsible for sustenance of the pathogen inside the host. With an aim to elucidate the catalytic mechanism and identification of intermediates involved, we describe in this study different crystal structures of GAPDH1 from methicillin-resistant S. aureus MRSA252 (SaGAPDH1) in apo and holo forms of wild type, thioacyl intermediate, and ternary complexes of active-site mutants with physiological substrate d-glyceraldehyde-3-phosphate (G3P) and coenzyme NAD(+). A new phosphate recognition site, "new P(i)" site, similar to that observed in GAPDH from Thermotoga maritima, is reported here, which is 3.40 A away from the "classical P(i)" site. Ternary complexes discussed are representatives of noncovalent Michaelis complexes in the ground state. d-G3P is bound to all the four subunits of C151S.NAD and C151G.NAD in more reactive hydrate (gem-di-ol) form. However, in C151S+H178N.NAD, the substrate is bound to two chains in aldehyde form and in gem-di-ol form to the other two. This work reports binding of d-G3P to the C151G mutant in an inverted manner for the very first time. The structure of the thiaocyl complex presented here is formed after the hydride transfer. The C3 phosphate of d-G3P is positioned at the "P(s)" site in the ternary complexes but at the "new P(i)" site in the thioacyl complex and C1-O1 bond points opposite to His178 disrupting the alignment between itself and NE2 of His178. A new conformation (Conformation I) of the 209-215 loop has also been identified, where the interaction between phosphate ion at the "new P(i)" site and conserved Gly212 is lost. Altogether, inferences drawn from the kinetic analyses and crystal structures suggest the "flip-flop" model proposed for the enzyme mechanism.

post to: CiteULike

Dimeric crystal structure of rabbit L-gulonate 3-dehydrogenase/lambda-crystallin: insights into the catalytic mechanism.

Publication Date: 2010 Sep 3 PMID: 20620150
Authors: Asada, Y. - Kuroishi, C. - Ukita, Y. - Sumii, R. - Endo, S. - Matsunaga, T. - Hara, A. - Kunishima, N.
Journal: J Mol Biol

l-Gulonate 3-dehydrogenase (GDH) is a bifunctional dimeric protein that functions not only as an NAD(+)-dependent enzyme in the uronate cycle but also as a taxon-specific lambda-crystallin in rabbit lens. Here we report the first crystal structure of GDH in both apo form and NADH-bound holo form. The GDH protomer consists of two structural domains: the N-terminal domain with a Rossmann fold and the C-terminal domain with a novel helical fold. In the N-terminal domain of the NADH-bound structure, we identified 11 coenzyme-binding residues and found 2 distinct side-chain conformers of Ser124, which is a putative coenzyme/substrate-binding residue. A structural comparison between apo form and holo form and a mutagenesis study with E97Q mutant suggest an induced-fit mechanism upon coenzyme binding; coenzyme binding induces a conformational change in the coenzyme-binding residues Glu97 and Ser124 to switch their activation state from resting to active, which is required for the subsequent substrate recruitment. Subunit dimerization is mediated by numerous intersubunit interactions, including 22 hydrogen bonds and 104 residue pairs of van der Waals interactions, of which those between two cognate C-terminal domains are predominant. From a structure/sequence comparison within GDH homologues, a much greater degree of interprotomer interactions (both polar and hydrophobic) in the rabbit GDH would contribute to its higher thermostability, which may be relevant to the other function of this enzyme as lambda-crystallin, a constitutive structural protein in rabbit lens. The present crystal structures and amino acid mutagenesis studies assigned the role of active-site residues: catalytic base for His145 and substrate binding for Ser124, Cys125, Asn196, and Arg231. Notably, Arg231 participates in substrate binding from the other subunit of the GDH dimer, indicating the functional significance of the dimeric state. Proper orientation of the substrate-binding residues for catalysis is likely to be maintained by an interprotomer hydrogen-bonding network of residues Asn196, Gln199, and Arg231, suggesting a network-based substrate recognition of GDH.

post to: CiteULike

The pathway of product release from the R state of aspartate transcarbamoylase.

Publication Date: 2010 Sep 3 PMID: 20620149
Authors: Mendes, K. R. - Kantrowitz, E. R.
Journal: J Mol Biol

The pathway of product release from the R state of aspartate transcarbamoylase (ATCase; EC 2.1.3.2, aspartate carbamoyltransferase) has been determined here by solving the crystal structure of Escherichia coli ATCase locked in the R quaternary structure by specific introduction of disulfide bonds. ATCase displays ordered substrate binding and product release, remaining in the R state until substrates are exhausted. The structure reported here represents ATCase in the R state bound to the final product molecule, phosphate. This structure has been difficult to obtain previously because the enzyme relaxes back to the T state after the substrates are exhausted. Hence, cocrystallizing the wild-type enzyme with phosphate results in a T-state structure. In this structure of the enzyme trapped in the R state with specific disulfide bonds, we observe two phosphate molecules per active site. The position of the first phosphate corresponds to the position of the phosphate of carbamoyl phosphate (CP) and the position of the phosphonate of N-phosphonacetyl-l-aspartate. However, the second, more weakly bound phosphate is bound in a positively charged pocket that is more accessible to the surface than the other phosphate. The second phosphate appears to be on the path that phosphate would have to take to exit the active site. Our results suggest that phosphate dissociation and CP binding can occur simultaneously and that the dissociation of phosphate may actually promote the binding of CP for more efficient catalysis.

post to: CiteULike

SMN, Gemin2 and Gemin3 associate with beta-actin mRNA in the cytoplasm of neuronal cells in vitro.

Publication Date: 2010 Sep 3 PMID: 20620147
Authors: Todd, A. G. - Morse, R. - Shaw, D. J. - McGinley, S. - Stebbings, H. - Young, P. J.
Journal: J Mol Biol

Childhood spinal muscular atrophy is caused by a reduced expression of the survival motor neuron (SMN) protein. SMN has been implicated in the axonal transport of beta-actin mRNA in both primary and transformed neuronal cell lines, and loss of this function could account, at least in part, for spinal muscular atrophy onset and pathological specificity. Here we have utilised a targeted screen to identify mRNA associated with SMN, Gemin2 and Gemin3 in the cytoplasm of a human neuroblastoma cell line, SHSY5Y. Importantly, we have provided the first direct evidence that beta-actin mRNA is present in SMN cytoplasmic complexes in SHSY5Y cells.

post to: CiteULike

Hydrophobic surface patches on LolA of Pseudomonas aeruginosa are essential for lipoprotein binding.

Publication Date: 2010 Sep 3 PMID: 20620146
Authors: Remans, K. - Pauwels, K. - van Ulsen, P. - Buts, L. - Cornelis, P. - Tommassen, J. - Savvides, S. N. - Decanniere, K. - Van Gelder, P.
Journal: J Mol Biol

Many lipoproteins reside in the outer membrane (OM) of Gram-negative bacteria, and their biogenesis is dependent on the Lol (localization of lipoproteins) system. The periplasmic chaperone LolA accepts OM-destined lipoproteins that are released from the inner membrane by the LolCDE complex and transfers them to the OM receptor LolB. The exact nature of the LolA-lipoprotein complex is still unknown. The crystal structure of Escherichia coli LolA features an open beta-barrel covered by alpha helices that together constitute a hydrophobic cavity, which would allow the binding of one acyl chain. However, OM lipoproteins contain three acyl chains, and the stoichiometry of the LolA-lipoprotein complex is 1:1. Here we present the crystal structure of Pseudomonas aeruginosa LolA that projects clear hydrophobic surface patches. Since these patches are large enough to accommodate acyl chains, their role in lipoprotein binding was investigated. Several LolA mutant proteins were created, and their functionality was assessed by studying their capacity to release lipoproteins produced in sphaeroplasts. Interruption of the largest hydrophobic patch completely destroyed the lipoprotein-releasing capacity of LolA, while interruption of smaller patches apparently reduced efficiency. Thus, the results show a new lipoprotein transport model that places (some of) the acyl chains on the hydrophobic surface patches.

post to: CiteULike

Glycosidic bond conformation preference plays a pivotal role in catalysis of RNA pseudouridylation: a combined simulation and structural study.

Publication Date: 2010 Sep 3 PMID: 20615421
Authors: Zhou, J. - Lv, C. - Liang, B. - Chen, M. - Yang, W. - Li, H.
Journal: J Mol Biol

The most abundant chemical modification on RNA is isomerization of uridine (or pseudouridylation) catalyzed by pseudouridine synthases. The catalytic mechanism of this essential process remains largely speculative, partly due to lack of knowledge of the pre-reactive state that is important to the identification of reactive chemical moieties. In the present study, we showed, using orthogonal space random-walk free-energy simulation, that the pre-reactive states of uridine and its reactive derivative 5-fluorouridine, bound to a ribonucleoprotein particle pseudouridine synthase, strongly prefer the syn glycosidic bond conformation, while that of the nonreactive 5-bromouridine-containing substrate is largely populated in the anti conformation state. A high-resolution crystal structure of the 5-bromouridine-containing substrate bound to the ribonucleoprotein particle pseudouridine synthase and enzyme activity assay confirmed the anti nonreactive conformation and provided the molecular basis for its confinement. The observed preference for the syn pre-reactive state by the enzyme-bound uridine may help to distinguish among currently proposed mechanisms.

post to: CiteULike

The critical roles of residues P235 and F236 of subunit A of the motor protein A-ATP synthase in P-loop formation and nucleotide binding.

Publication Date: 2010 Sep 3 PMID: 20615420
Authors: Kumar, A. - Manimekalai, M. S. - Balakrishna, A. M. - Priya, R. - Biukovic, G. - Jeyakanthan, J. - Gruber, G.
Journal: J Mol Biol

The mutants P235A and F236A have been generated and their crystal structure was determined to resolutions of 2.38 and 2.35 A, respectively, in order to understand the residues involved in the formation of the novel arched P-loop of subunit A of the A-ATP synthase from Pyrococcus horikoshii OT3. Both the structures show unique, altered conformations for the P-loop. Comparison with the previously solved wild type and P-loop mutant S238A structures of subunit A showed that the P-loop conformation for these two novel mutants occupy intermediate positions, with the wild type fully arched and the well-relaxed S238A mutant structures taking the extreme positions. Even though the deviation is similar for both mutants, the curvature of the P-loop faces the opposite direction. Deviations in the GER-loop, lying above the P-loop, are similar for both mutants, but in F236A, it moves towards the P-loop by around 2 A. The curvature of the loop region V392-V410, located directly behind the P-loop, moves close by 3.6 A towards the P-loop in the F236A structure and away by 2.5 A in the P235A structure. Two major deviations were observed in the P235A mutant, which are not identified in any of the subunit A structures analyzed so far, one being a wide movement of the N-terminal loop region (R90-P110) making a rotation of 80 degrees and the other being rigid-body rotation of the C-terminal helices from Q520-A588 by around 4 degrees upwards. Taken together, the data presented demonstrate the concerted effects of the critical residues P235A, F236, and S238 in the unique P-loop conformation of the A-ATP synthases.

post to: CiteULike

Specificity for homooligomer versus heterooligomer formation in integrin transmembrane helices.

Publication Date: 2010 Sep 3 PMID: 20615419
Authors: Zhu, H. - Metcalf, D. G. - Streu, C. N. - Billings, P. C. - Degrado, W. F. - Bennett, J. S.
Journal: J Mol Biol

Transmembrane (TM) helices engage in homomeric and heteromeric interactions that play essential roles in the folding and assembly of TM proteins. However, features that explain their propensity to interact homomerically or heteromerically and determine the strength of these interactions are poorly understood. Integrins provide an ideal model system for addressing these questions because the TM helices of full-length integrins interact heteromerically when integrins are inactive, but isolated TM helices are also able to form homodimers or homooligomers in micelles and bacterial membranes. We sought to determine the features defining specificity for homointeractions versus heterointeractions by conducting a comprehensive comparison of the homomeric and heteromeric interactions of integrin alphaIIbbeta3 TM helices in biological membranes. Using the TOXCAT assay, we found that residues V700, M701, A703, I704, L705, G708, L709, L712, and L713, which are located on the same face of the beta3 helix, mediate homodimer formation. We then characterized the beta3 heterodimer by measuring the ability of beta3 helix mutations to cause ligand binding to alphaIIbbeta3. We found that mutating V696, L697, V700, M701, A703. I704, L705, G708, L712, and L713, but not the small residue-X(3)-small residue motif S699-X(3)-A703, caused constitutive alphaIIbbeta3 activation, as well as persistent focal adhesion kinase phosphorylation dependent on alphaIIbbeta3 activation. Because alphaIIb and beta3 use the same face of their respective TM helices for homomeric and heteromeric interactions, the interacting surface on each has an intrinsic "stickiness" predisposing towards helix-helix interactions in membranes. The residues responsible for heterodimer formation comprise a network of interdigitated side chains with considerable geometric complementarity; mutations along this interface invariably destabilize heterodimer formation. By contrast, residues responsible for homomeric interactions are dispersed over a wider surface. While most mutations of these residues are destabilizing, some stabilized homooligomer formation. We conclude that the alphaIIbbeta3 TM heterodimer shows the hallmark of finely tuned heterodimeric interaction, while homomeric interaction is less specific.

post to: CiteULike

Structure-based annotation of a novel sugar isomerase from the pathogenic E. coli O157:H7.

Publication Date: 2010 Sep 3 PMID: 20615418
Authors: van Staalduinen, L. M. - Park, C. S. - Yeom, S. J. - Adams-Cioaba, M. A. - Oh, D. K. - Jia, Z.
Journal: J Mol Biol

Prokaryotes can use a variety of sugars as carbon sources in order to provide a selective survival advantage. The gene z5688 found in the pathogenic Escherichia coli O157:H7 encodes a "hypothetical" protein of unknown function. Sequence analysis identified the gene product as a putative member of the cupin superfamily of proteins, but no other functional information was known. We have determined the crystal structure of the Z5688 protein at 1.6 A resolution and identified the protein as a novel E. coli sugar isomerase (EcSI) through overall fold analysis and secondary-structure matching. Extensive substrate screening revealed that EcSI is capable of acting on d-lyxose and d-mannose. The complex structure of EcSI with fructose allowed the identification of key active-site residues, and mutagenesis confirmed their importance. The structure of EcSI also suggested a novel mechanism for substrate binding and product release in a cupin sugar isomerase. Supplementation of a nonpathogenic E. coli strain with EcSI enabled cell growth on the rare pentose d-lyxose.

post to: CiteULike

Charge-rich regions modulate the anti-aggregation activity of Hsp90.

Publication Date: 2010 Sep 3 PMID: 20615417
Authors: Wayne, N. - Bolon, D. N.
Journal: J Mol Biol

Protein aggregation can have dramatic effects on cellular function and plays a causative role in many human diseases. In all cells, molecular chaperones bind to aggregation-prone proteins and hinder aggregation. The ability of a protein to resist aggregation and remain soluble in aqueous solution is linked to the physical properties of the protein. Numerous physical studies demonstrate that charged atoms favor solubility. We note that many molecular chaperones possess a substantial negative charge that may allow them to impart solubility on aggregation-prone proteins. Hsp90 is one such negatively charged molecular chaperone. The charge on Hsp90 is largely concentrated in two highly acidic regions. To investigate the relationship between chaperone charge and protein solubility, we deleted these charge-rich regions and analyzed the resulting Hsp90 constructs for anti-aggregation activity. We found that deletion of both charge-rich regions dramatically impaired Hsp90 anti-aggregation activity. The anti-aggregation role of the deleted charge-rich regions could be due to net charge or sequence-specific features. To distinguish these possibilities, we attached an acid-rich region with a distinct amino acid sequence to our double-deleted Hsp90 construct. This charge rescue construct displayed effective anti-aggregation activity indicating that the net charge of Hsp90 contributes to its anti-aggregation activity.

post to: CiteULike

Both the p33 and p55 subunits of the Helicobacter pylori VacA toxin are targeted to mammalian mitochondria.

Publication Date: 2010 Sep 3 PMID: 20615415
Authors: Foo, J. H. - Culvenor, J. G. - Ferrero, R. L. - Kwok, T. - Lithgow, T. - Gabriel, K.
Journal: J Mol Biol

Helicobacter pylori infection causes peptic ulcers and gastric cancer. A major toxin secreted by H. pylori is the bipartite vacuolating cytotoxin A, VacA. The toxin is believed to enter host cells as two subunits: the p55 subunit (55 kDa) and the p33 subunit (33 kDa). At the biochemical level, it has been shown that VacA forms through the assembly of large multimeric pores composed of both the p33 subunit and the p55 subunit in biological membranes. One of the major target organelles of VacA is the mitochondria. Since only the p33 subunit has been reported to be translocated into mitochondria and the p55 subunit is not imported, it has been contentious as to whether VacA assembles into pores in a mitochondrial membrane. Here we show the p55 protein is imported into the mitochondria along with the p33 protein subunit. The p33 subunit integrally associates with the mitochondrial inner membrane, and both the p33 subunit and the p55 subunit are exposed to the mitochondrial intermembrane space. Their colocalization suggests that they could reassemble and form a pore in the inner mitochondrial membrane.

post to: CiteULike

Tyr39 of ran preserves the Ran.GTP gradient by inhibiting GTP hydrolysis.

Publication Date: 2010 Aug 6 PMID: 20609434
Authors: Brucker, S. - Gerwert, K. - Kotting, C.
Journal: J Mol Biol

Ran is a member of the superfamily of small GTPases, which cycle between a GTP-bound "on" and a GDP-bound "off" state. Ran regulates nuclear transport. In order to maintain a gradient of excess Ran.GTP within the nucleoplasm and excess Ran.GDP within the cytoplasm, the hydrolysis of Ran.GTP in the nucleoplasm should be prevented, whereas in the cytoplasm, hydrolysis is catalyzed by Ran.GAP (GTPase-activating protein). In this article, we investigate the GTPase reaction of Ran in complex with its binding protein Ran-binding protein 1 by time-resolved Fourier transform infrared spectroscopy: We show that the slowdown of the intrinsic hydrolysis of RanGTP is accomplished by tyrosine 39, which is probably misplacing the attacking water. We monitored the interaction of Ran with RanGAP, which reveals two reactions steps. By isotopic labeling of Ran and RanGAP, we were able to assign the first step to a small conformational change within the catalytic site. The following bond breakage is the rate-limiting step of hydrolysis. An intermediate of protein-bound phosphate as found for Ras or Rap systems is kinetically unresolved. This demonstrates that despite the structural similarity among the G-domain of the GTPases, different reaction mechanisms are utilized.

MeSH Categories: Adsorption, Animals, Catalytic Domain, Eukaryotic Cells/metabolism, GTP Phosphohydrolases/*antagonists &, inhibitors/chemistry/metabolism/physiology, GTPase-Activating Proteins/chemistry/metabolism, Guanosine Triphosphate/*metabolism, Humans, Hydrolysis, Models, Biological, Models, Molecular, Protein Conformation, Protein Transport, Tyrosine/chemistry/*physiology, ran GTP-Binding Protein/antagonists &, inhibitors/*chemistry/metabolism/*physiology

post to: CiteULike

A monomeric photoconvertible fluorescent protein for imaging of dynamic protein localization.

Publication Date: 2010 Sep 3 PMID: 20603133
Authors: Hoi, H. - Shaner, N. C. - Davidson, M. W. - Cairo, C. W. - Wang, J. - Campbell, R. E.
Journal: J Mol Biol

The use of green-to-red photoconvertible fluorescent proteins (FPs) enables researchers to highlight a subcellular population of a fusion protein of interest and to image its dynamics in live cells. In an effort to enrich the arsenal of photoconvertible FPs and to overcome the limitations imposed by the oligomeric structure of natural photoconvertible FPs, we designed and optimized a new monomeric photoconvertible FP. Using monomeric versions of Clavularia sp. cyan FP as template, we employed sequence-alignment-guided design to create a chromophore environment analogous to that shared by known photoconvertible FPs. The designed gene was synthesized and, when expressed in Escherichia coli, found to produce green fluorescent colonies that gradually switched to red after exposure to white light. We subjected this first-generation FP [named mClavGR1 (monomeric Clavularia-derived green-to-red photoconvertible 1)] to a combination of random and targeted mutageneses and screened libraries for efficient photoconversion using a custom-built system for illuminating a 10-cm Petri plate with 405-nm light. Following more than 15 rounds of library creation and screening, we settled on an optimized version, known as mClavGR2, that has eight mutations relative to mClavGR1. Key improvements of mClavGR2 relative to mClavGR1 include a 1.4-fold brighter red species, 1.8-fold higher photoconversion contrast, and dramatically improved chromophore maturation in E. coli. The monomeric status of mClavGR2 has been demonstrated by gel-filtration chromatography and the functional expression of a variety of mClavGR2 chimeras in mammalian cells. Furthermore, we have exploited mClavGR2 to determine the diffusion kinetics of the membrane protein intercellular adhesion molecule 1 both when the membrane is in contact with a T-lymphocyte expressing leukocyte-function-associated antigen 1 and when it is not. These experiments clearly establish that mClavGR2 is well suited for rapid photoconversion of protein subpopulations and subsequent tracking of dynamic changes in localization in living cells.

post to: CiteULike

Nonmuscle myosin-dependent synthesis of type I collagen.

Publication Date: 2010 Aug 27 PMID: 20603131
Authors: Cai, L. - Fritz, D. - Stefanovic, L. - Stefanovic, B.
Journal: J Mol Biol

Type I collagen, synthesized in all tissues as the heterotrimer of two alpha1(I) polypeptides and one alpha2(I) polypeptide, is the most abundant protein in the human body. Here we show that intact nonmuscle myosin filaments are required for the synthesis of heterotrimeric type I collagen. Conserved 5' stem-loop in collagen alpha1(I) and alpha2(I) mRNAs binds the RNA-binding protein LARP6. LARP6 interacts with nonmuscle myosin through its C-terminal domain and associates collagen mRNAs with the filaments. Dissociation of nonmuscle myosin filaments results in secretion of collagen alpha1(I) homotrimer, diminished intracellular colocalization of collagen alpha1(I) and alpha2(I) polypeptides (required for folding of the heterotrimer), and their increased intracellular degradation. Inhibition of the motor function of myosin has similar collagen-specific effects, while disruption of actin filaments has a general effect on protein secretion. Nonmuscle myosin copurifies with polysomes, and there is a subset of polysomes involved in myosin-dependent translation of collagen mRNAs. These results indicate that association of collagen mRNAs with nonmuscle myosin filaments is necessary to coordinately synthesize collagen alpha1(I) and alpha2(I) polypeptides. We postulate that LARP6/myosin-dependent mechanism regulates the synthesis of heterotrimeric type I collagen by coordinating the translation of collagen mRNAs.

MeSH Categories: Animals, Cells, Cultured, Collagen Type I/*biosynthesis, Humans, Mice, Mice, Knockout, Myosins/*metabolism, Polyribosomes/metabolism, RNA, Messenger/metabolism, RNA-Binding Proteins/genetics/metabolism

post to: CiteULike

Novel aspects of the acid response network of E. coli K-12 are revealed by a study of transcriptional dynamics.

Publication Date: 2010 Sep 3 PMID: 20603130
Authors: Burton, N. A. - Johnson, M. D. - Antczak, P. - Robinson, A. - Lund, P. A.
Journal: J Mol Biol

Understanding gene regulation and its adaptive significance requires not only a detailed knowledge of individual molecular interactions that give rise to changes in gene expression but also an overview of complete genetic networks and the ways in which components within them interact. Increasingly, such studies are being done using luminescent or fluorescent reporter proteins that enable monitoring of gene expression dynamics in real time, particularly during changes in expression. We show here that such an approach is valid for dissecting the responses of the AR2 or GAD network of Escherichia coli K-12 to changes in pH, which is one of the most complex networks known in E. coli. In addition to confirming several regulatory interactions that have been revealed by previous studies, this approach has identified new components in this system that lead to complex dynamics of gene expression following a drop in pH, including an auto-regulatory loop involving the YdeO activator protein and novel roles for the PhoP protein.

post to: CiteULike

Bacillus anthracis surface-layer proteins assemble by binding to the secondary cell wall polysaccharide in a manner that requires csaB and tagO.

Publication Date: 2010 Sep 3 PMID: 20603129
Authors: Kern, J. - Ryan, C. - Faull, K. - Schneewind, O.
Journal: J Mol Biol

Bacillus anthracis, the causative agent of anthrax, requires surface (S)-layer proteins for the pathogenesis of infection. Previous work characterized S-layer protein binding via the surface layer homology domain to a pyruvylated carbohydrate in the envelope of vegetative forms. The molecular identity of this carbohydrate and the mechanism of its display in the bacterial envelope are still unknown. Analyzing acid-solubilized, purified carbohydrates by mass spectrometry and NMR spectroscopy, we identify secondary cell wall polysaccharide (SCWP) as the ligand of S-layer proteins. In agreement with the model that surface layer homology domains bind to pyruvylated carbohydrate, SCWP was observed to be linked to pyruvate in a manner requiring csaB, the only structural gene known to be required for S-layer assembly. B. anthracis does not elaborate wall teichoic acids; however, its genome harbors tagO and tagA, genes responsible for the synthesis of the linkage unit that tethers teichoic acids to the peptidoglycan layer. The tagO gene appears essential for B. anthracis growth and complements the tagO mutant phenotypes of staphylococci. Tunicamycin-mediated inhibition of TagO resulted in deformed, S-layer-deficient bacilli. Together, these results suggest that tagO-mediated assembly of linkage units tethers pyruvylated SCWP to the B. anthracis envelope, thereby enabling S-layer assembly and providing for the pathogenesis of anthrax infections.

post to: CiteULike

Abeta(1-40) forms five distinct amyloid structures whose beta-sheet contents and fibril stabilities are correlated.

Publication Date: 2010 Aug 20 PMID: 20600131
Authors: Kodali, R. - Williams, A. D. - Chemuru, S. - Wetzel, R.
Journal: J Mol Biol

The ability of a single polypeptide sequence to grow into multiple stable amyloid fibrils sets these aggregates apart from most native globular proteins. The existence of multiple amyloid forms is the basis for strain effects in yeast prion biology, and might contribute to variations in Alzheimer's disease pathology. However, the structural basis for amyloid polymorphism is poorly understood. We report here five structurally distinct fibrillar aggregates of the Alzheimer's plaque peptide Abeta(1-40), as well as a non-fibrillar aggregate induced by Zn(2+). Each of these conformational forms exhibits a unique profile of physical properties, and all the fibrillar forms breed true in elongation reactions under a common set of growth conditions. Consistent with their defining cross-beta structure, we find that in this series the amyloid fibrils containing more extensive beta-sheet exhibit greater stability. At the same time, side chain packing outside of the beta-sheet regions contributes to stability, and to differences of stability between polymorphic forms. Stability comparison is facilitated by the unique feature that the free energy of the monomer (equivalent to the unfolded state in a protein folding reaction) does not vary, and hence can be ignored, in the comparison of DeltaG degrees of elongation values for each polymorphic fibril obtained under a single set of conditions.

post to: CiteULike

Asymmetric dimeric structure of ferredoxin-NAD(P)+ oxidoreductase from the green sulfur bacterium Chlorobaculum tepidum: implications for binding ferredoxin and NADP+.

Publication Date: 2010 Aug 20 PMID: 20600130
Authors: Muraki, N. - Seo, D. - Shiba, T. - Sakurai, T. - Kurisu, G.
Journal: J Mol Biol

Ferredoxin-NAD(P)(+) oxidoreductase (FNR) catalyzes the reduction of NAD(P)(+) to NAD(P)H with the reduced ferredoxin (Fd) during the final step of the photosynthetic electron transport chain. FNR from the green sulfur bacterium Chlorobaculum tepidum is functionally analogous to plant-type FNR but shares a structural homology to NADPH-dependent thioredoxin reductase (TrxR). Here, we report the crystal structure of C. tepidum FNR to 2.4 A resolution, which reveals a unique structure-function relationship. C. tepidum FNR consists of two functional domains for binding FAD and NAD(P)H that form a homodimer in which the domains are arranged asymmetrically. One NAD(P)H domain is present as the open form, the other with the equivalent NAD(P)H domain as the relatively closed form. We used site-directed mutagenesis on the hinge region connecting the two domains in order to investigate the importance of the flexible hinge. The asymmetry of the NAD(P)H domain and the comparison with TrxR suggested that the hinge motion might be involved in pyridine nucleotide binding and binding of Fd. Surprisingly, the crystal structure revealed an additional C-terminal sub-domain that tethers one protomer and interacts with the other protomer by pi-pi stacking of Phe337 and the isoalloxazine ring of FAD. The position of this stacking Phe337 is almost identical with both of the conserved C-terminal Tyr residues of plant-type FNR and the active site dithiol of TrxR, implying a unique structural basis for enzymatic reaction of C. tepidum FNR.

post to: CiteULike

Structure and reactivity of Bacillus subtilis MenD catalyzing the first committed step in menaquinone biosynthesis.

Publication Date: 2010 Aug 13 PMID: 20600129
Authors: Dawson, A. - Chen, M. - Fyfe, P. K. - Guo, Z. - Hunter, W. N.
Journal: J Mol Biol

The first committed step in the classical biosynthetic route to menaquinone (vitamin K(2)) is a Stetter-like conjugate addition of alpha-ketoglutarate with isochorismate. This reaction is catalyzed by the thiamine diphosphate and metal-ion-dependent 2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexadiene-1-carboxylate synthase (MenD). The medium-resolution (2.35 A) crystal structure of Bacillus subtilis MenD with cofactor and Mn(2+) has been determined. Based on structure-sequence comparisons and modeling, a two-stage mechanism that is primarily driven by the chemical properties of the cofactor is proposed. Hypotheses for the molecular determinants of substrate recognition were formulated. Five basic residues (Arg32, Arg106, Arg409, Arg428, and Lys299) are postulated to interact with carboxylate and hydroxyl groups to align substrates for catalysis in combination with a cluster of non-polar residues (Ile489, Phe490, and Leu493) on one side of the active site. The powerful combination of site-directed mutagenesis, where each of the eight residues is replaced by alanine, and steady-state kinetic measurements has been exploited to address these hypotheses. Arg409 plays a significant role in binding both substrates while Arg428 contributes mainly to binding of alpha-ketoglutarate. Arg32 and in particular Arg106 are critical for recognition of isochorismate. Mutagenesis of Phe490 and Ile489 has the most profound influence on catalytic efficiency, indicating that these two residues are important for binding of isochorismate and for stabilizing the cofactor position. These data allow for a detailed description of the structure-reactivity relationship that governs MenD function and refinement of the model for the catalytic intermediate that supports the Stetter-like conjugate addition.

MeSH Categories: Amino Acid Sequence, Amino Acid Substitution, Bacillus subtilis/*enzymology/genetics, Bacterial Proteins/*chemistry/genetics/*metabolism, Base Sequence, Catalysis, Catalytic Domain/genetics, Crystallography, X-Ray, DNA, Bacterial/genetics, Escherichia coli Proteins/chemistry/genetics/metabolism, Kinetics, Molecular Sequence Data, Mutagenesis, Site-Directed, Protein Conformation, Protein Structure, Quaternary, Pyruvate Oxidase/*chemistry/genetics/*metabolism, Recombinant Proteins/chemistry/genetics/metabolism, Sequence Homology, Amino Acid, Sesquiterpenes, Species Specificity, Structural Homology, Protein, Substrate Specificity, Vitamin K 2/*metabolism

post to: CiteULike

Mechanism of DNA recognition by the restriction enzyme EcoRV.

Publication Date: 2010 Aug 20 PMID: 20600128
Authors: Zahran, M. - Daidone, I. - Smith, J. C. - Imhof, P.
Journal: J Mol Biol

EcoRV, a restriction enzyme in Escherichia coli, destroys invading foreign DNA by cleaving it at the center step of a GATATC sequence. In the EcoRV-cognate DNA crystallographic complex, a sharp kink of 50 degrees has been found at the center base-pair step (TA). Here, we examine the interplay between the intrinsic propensity of the cognate sequence to kink and the induction by the enzyme by performing all-atom molecular dynamics simulations of EcoRV unbound and interacting with three DNA sequences: the cognate sequence, GATATC (TA); the non-cognate sequence, GAATTC (AT); and with the cognate sequence methylated on the first adenine GA(CH(3))TATC (TA-CH(3)). In the unbound EcoRV, the cleft between the two C-terminal subdomains is found to be open. Binding to AT narrows the cleft and forms a partially bound state. However, the intrinsic bending propensity of AT is insufficient to allow tight binding. In contrast, the cognate TA sequence is easier to bend, allowing specific, high-occupancy hydrogen bonds to form in the complex. The absence of cleavage for this methylated sequence is found to arise from the loss of specific hydrogen bonds between the first adenine of the recognition sequence and Asn185. On the basis of the results, we suggest a three-step recognition mechanism. In the first step, EcoRV, in an open conformation, binds to the DNA at a random sequence and slides along it. In the second step, when the two outer base pairs, GAxxTC, are recognized, the R loops of the protein become more ordered, forming strong hydrogen-bonding interactions, resulting in a partially bound EcoRV-DNA complex. In the third step, the flexibility of the center base pair is probed, and in the case of the full cognate sequence the DNA bends, the complex strengthens and the protein and DNA interact more closely, allowing cleavage.

post to: CiteULike

Response of the bacteriophage T4 replisome to noncoding lesions and regression of a stalled replication fork.

Publication Date: 2010 Sep 3 PMID: 20600127
Authors: Nelson, S. W. - Benkovic, S. J.
Journal: J Mol Biol

DNA is constantly damaged by endogenous and exogenous agents. The resulting DNA lesions have the potential to halt the progression of the replisome, possibly leading to replication fork collapse. Here, we examine the effect of a noncoding DNA lesion in either leading strand template or lagging strand template on the bacteriophage T4 replisome. A damaged base in the lagging strand template does not affect the progression of the replication fork. Instead, the stalled lagging strand polymerase recycles from the lesion and initiates the synthesis of a new Okazaki fragment upstream of the damaged base. In contrast, when the replisome encounters a blocking lesion in the leading strand template, the replication fork only travels approximately 1 kb beyond the point of the DNA lesion before complete replication fork collapse. The primosome and the lagging strand polymerase remain active during this period, and an Okazaki fragment is synthesized beyond the point of the leading strand lesion. There is no evidence for a new priming event on the leading strand template. Instead, the DNA structure that is produced by the stalled replication fork is a substrate for the DNA repair helicase UvsW. UvsW catalyzes the regression of a stalled replication fork into a "chicken-foot" structure that has been postulated to be an intermediate in an error-free lesion bypass pathway.

post to: CiteULike

Interactions of the melanocortin-4 receptor with the peptide agonist NDP-MSH.

Publication Date: 2010 Aug 20 PMID: 20600126
Authors: Chapman, K. L. - Kinsella, G. K. - Cox, A. - Donnelly, D. - Findlay, J. B.
Journal: J Mol Biol

Melanocortin-4 receptor (MC4R) has an important regulatory role in energy homeostasis and food intake. Peptide agonists of the MC4R are characterized by the conserved sequence His(6)-Phe(7)-Arg(8)-Trp(9), which is crucial for their interaction with the receptor. This investigation utilized the covalent attachment approach to identify receptor residues in close proximity to the bound ligand [Nle(4),D-Phe(7)]melanocyte-stimulating hormone (NDP-MSH), thereby differentiating between residues directly involved in ligand binding and those mutations that compromise ligand binding by inducing conformational changes in the receptor. Also, recent X-ray structures of G-protein-coupled receptors were utilized to refine a model of human MC4R in the active state (R(*)), which was used to generate a better understanding of the binding mode of the ligand NDP-MSH at the atomic level. The mutation of residues in the human MC4R--such as Leu106 of extracellular loop 1, and Asp122, Ile125, and Asp126 of transmembrane (TM) helix 3, His264 (TM6), and Met292 (TM7)--to Cys residues produced definitive indications of proximity to the side chains of residues in the core region of the peptide ligand. Of particular interest was the contact between D-Phe(7) on the ligand and Ile125 of TM3 on the MC4R. Additionally, Met292 (TM7) equivalent to Lys(7.45) (Ballesteros numbering scheme) involved in covalently attaching retinal in rhodopsin is shown to be in close proximity to Trp(9). For the first time, the interactions between the terminal regions of NDP-MSH and the receptor are described. The amino-terminus appears to be adjacent to a series of hydrophilic residues with novel interactions at Cys196 (TM5) and Asp189 (extracellular loop 2). These interactions are reminiscent of sequential ligand binding exhibited by the beta(2)-adrenergic receptor, with the former interaction being equivalent to the known interaction involving Ser204 of the beta(2)-adrenergic receptor.

post to: CiteULike

Structural basis for adenylate kinase activity in ABC ATPases.

Publication Date: 2010 Aug 13 PMID: 20600125
Authors: Lammens, A. - Hopfner, K. P.
Journal: J Mol Biol

ATP-binding cassette (ABC) enzymes are involved in diverse biological processes ranging from transmembrane transport to chromosome cohesion and DNA repair. They typically use ATP hydrolysis to conduct energy-dependent biological reactions. However, the cystic fibrosis transmembrane conductance regulator and the DNA repair protein Rad50 can also catalyze the adenylate kinase reaction (ATP+AMP<-->2ADP). To clarify and provide a mechanistic basis for the adenylate kinase activity of ABC enzymes, we report the crystal structure of the nucleotide-binding domain of the Pyrococcus furiosus structural maintenance of chromosome protein (pfSMC(nbd)) in complex with the adenylate kinase inhibitor P(1),P(5)-di(adenosine-5')pentaphosphate. We show that pfSMC(nbd) possesses reverse adenylate kinase activity. Our results suggest that in adenylate kinase reactions, ATP binds to its canonical binding site while AMP binds to the Q-loop glutamine and a hydration water of the Mg(2+) ion. Furthermore, mutational analysis indicates that adenylate kinase reaction occurs in the engaged pfSMC(nbd) dimer and requires the Signature motif for phosphate transfer. Our results explain how ATP hydrolysis and adenylate kinase reactions can be catalyzed by the same functional motifs within the structural framework of ABC enzymes. Thus, adenylate kinase activity is likely to be a latent activity in many ABC enzymes.

MeSH Categories: ATP-Binding Cassette Transporters/*chemistry/genetics/*metabolism, Adenine Nucleotides/metabolism, Adenosine Triphosphatases/*chemistry/genetics/*metabolism, Adenylate Kinase/*chemistry/genetics/*metabolism, Archaeal Proteins/chemistry/genetics/metabolism, Binding Sites, Crystallography, X-Ray, Cystic Fibrosis Transmembrane Conductance Regulator/chemistry/metabolism, DNA Repair Enzymes/chemistry/genetics/metabolism, Models, Molecular, Protein Conformation, Pyrococcus furiosus/genetics/metabolism

post to: CiteULike

Crystal structures of Bacillus subtilis Lon protease.

Publication Date: 2010 Aug 27 PMID: 20600124
Authors: Duman, R. E. - Lowe, J.
Journal: J Mol Biol

Lon ATP-dependent proteases are key components of the protein quality control systems of bacterial cells and eukaryotic organelles. Eubacterial Lon proteases contain an N-terminal domain, an ATPase domain, and a protease domain, all in one polypeptide chain. The N-terminal domain is thought to be involved in substrate recognition, the ATPase domain in substrate unfolding and translocation into the protease chamber, and the protease domain in the hydrolysis of polypeptides into small peptide fragments. Like other AAA+ ATPases and self-compartmentalising proteases, Lon functions as an oligomeric complex, although the subunit stoichiometry is currently unclear. Here, we present crystal structures of truncated versions of Lon protease from Bacillus subtilis (BsLon), which reveal previously unknown architectural features of Lon complexes. Our analytical ultracentrifugation and electron microscopy show different oligomerisation of Lon proteases from two different bacterial species, Aquifex aeolicus and B. subtilis. The structure of BsLon-AP shows a hexameric complex consisting of a small part of the N-terminal domain, the ATPase, and protease domains. The structure shows the approximate arrangement of the three functional domains of Lon. It also reveals a resemblance between the architecture of Lon proteases and the bacterial proteasome-like protease HslUV. Our second structure, BsLon-N, represents the first 209 amino acids of the N-terminal domain of BsLon and consists of a globular domain, similar in structure to the E. coli Lon N-terminal domain, and an additional four-helix bundle, which is part of a predicted coiled-coil region. An unexpected dimeric interaction between BsLon-N monomers reveals the possibility that Lon complexes may be stabilised by coiled-coil interactions between neighbouring N-terminal domains. Together, BsLon-N and BsLon-AP are 36 amino acids short of offering a complete picture of a full-length Lon protease.

MeSH Categories: Bacillus subtilis/*enzymology, Bacterial Proteins/*chemistry/metabolism, Crystallography, X-Ray, Microscopy, Electron, Models, Molecular, Protease La/*chemistry/metabolism, Protein Conformation, Protein Multimerization, Protein Structure, Tertiary

post to: CiteULike

Conformational dynamics in the selectivity filter of KcsA in response to potassium ion concentration.

Publication Date: 2010 Aug 13 PMID: 20600123
Authors: Bhate, M. P. - Wylie, B. J. - Tian, L. - McDermott, A. E.
Journal: J Mol Biol

Conformational change in the selectivity filter of KcsA as a function of ambient potassium concentration is studied with solid-state NMR. This highly conserved region of the protein is known to chelate potassium ions selectively. We report solid-state NMR chemical shift fingerprints of two distinct conformations of the selectivity filter; significant changes are observed in the chemical shifts of key residues in the filter as the potassium ion concentration is changed from 50 mM to 1 muM. Potassium ion titration studies reveal that the site-specific K(d) for K(+) binding at the key pore residue Val76 is on the order of approximately 7 muM and that a relatively high sample hydration is necessary to observe the low-K(+) conformer. Simultaneous detection of both conformers at low ambient potassium concentration suggests that the high-K(+) and low-K(+) states are in slow exchange on the NMR timescale (k(ex)<500 s(-)(1)). The slow rate and tight binding for evacuating both inner sites simultaneously differ from prior observations in detergent in solution, but agree well with measurements by electrophysiology and appear to result from our use of a hydrated bilayer environment. These observations strongly support a common assumption that the low-K(+) state is not involved in ion transmission, and that during transmission one of the two inner sites is always occupied. On the other hand, these kinetic and thermodynamic characteristics of the evacuation of the inner sites certainly could be compatible with participation in a control mechanism at low ion concentration such as C-type inactivation, a process that is coupled to activation and involves closing of the outer mouth of the channel.

MeSH Categories: Bacterial Proteins/*chemistry/genetics/*metabolism, Binding Sites, Kinetics, Models, Molecular, Molecular Dynamics Simulation, Nuclear Magnetic Resonance, Biomolecular, Potassium/*metabolism, Potassium Channels/*chemistry/genetics/*metabolism, Protein Conformation, Recombinant Proteins/chemistry/genetics/metabolism, Spectrum Analysis, Streptomyces lividans/genetics/metabolism, Water/chemistry

post to: CiteULike

Novel protein-protein contacts facilitate mRNA 3'-processing signal recognition by Rna15 and Hrp1.

Publication Date: 2010 Aug 20 PMID: 20600122
Authors: Leeper, T. C. - Qu, X. - Lu, C. - Moore, C. - Varani, G.
Journal: J Mol Biol

Precise 3'-end processing of mRNA is essential for correct gene expression, yet in yeast, 3'-processing signals consist of multiple ambiguous sequence elements. Two neighboring elements upstream of the cleavage site are particularly important for the accuracy (positioning element) and efficiency (efficiency element) of 3'-processing and are recognized by the RNA-binding proteins Rna15 and Hrp1, respectively. In vivo, these interactions are strengthened by the scaffolding protein Rna14 that stabilizes their association. The NMR structure of the 34 -kDa ternary complex of the RNA recognition motif (RRM) domains of Hrp1 and Rna15 bound to this pair of RNA elements was determined by residual dipolar coupling and paramagnetic relaxation experiments. It reveals how each of the proteins binds to RNA and introduces a novel class of protein-protein contact in regions of previously unknown function. These interdomain contacts had previously been overlooked in other multi-RRM structures, although a careful analysis suggests that they may be frequently present. Mutations in the regions of these contacts disrupt 3'-end processing, suggesting that they may structurally organize the ribonucleoprotein complexes responsible for RNA processing.

post to: CiteULike

A novel quasi-species of glutathione transferase with high activity towards naturally occurring isothiocyanates evolves from promiscuous low-activity variants.

Publication Date: 2010 Aug 20 PMID: 20600121
Authors: Runarsdottir, A. - Mannervik, B.
Journal: J Mol Biol

Glutathione transferases (GSTs) are known as promiscuous enzymes capable of catalyzing the conjugation of glutathione with a broad range of electrophilic substrates. A previous study based on recombinant chimeras derived from human GST M1-1 and GST M2-2 demonstrated the formation of a subset of F1 generation GSTs, which had lost high activity with substrates distinguishing parental enzymes. In the present study, the members of this subset were recombined by DNA shuffling to produce an F2 generation of GSTs. Screening of 930 bacterial clones demonstrated that 83% of recombinant enzyme variants were active with at least one of three alternative substrates: phenethyl isothiocyanate (PEITC), 1-chloro-2,4-dinitrobenzene, or p-nitrophenyl acetate. The majority had similar low activity as the parental GSTs in the F1 generation. However, 17 novel enzymes displayed high activity with PEITC. Half of these enzymes were similar to GST M1-1, which also has high activity with the same substrate, and all of these GSTs featured Tyr116/Ser210 in the active site. This group of F2 variants apparently had reverted to the GST M1-1 type. A second group of F2 variants with high PEITC activity was characterized by His116 in the active site. This category represented a new variety of GSTs, which demonstrated higher selectivity for isothiocyanate substrates than the GST M1-1 type. The different groups of GSTs can be considered as distinct molecular quasi-species, each of which comprises variant amino acid sequences. The quasi-species are structurally distinguished by active-site residues that govern their substrate selectivities. Clearly, minimal alterations of the active site can generate enzymes with highly distinctive functional properties.

post to: CiteULike

Mutational studies uncover non-native structure in the dimeric kinetic intermediate of the H2A-H2B heterodimer.

Publication Date: 2010 Aug 20 PMID: 20600120
Authors: Stump, M. R. - Gloss, L. M.
Journal: J Mol Biol

The folding pathway of the histone H2A-H2B heterodimer minimally includes an on-pathway, dimeric, burst-phase intermediate, I(2). The partially folded H2A and H2B monomers populated at equilibrium were characterized as potential monomeric kinetic intermediates. Folding kinetics were compared for initiation from isolated, folded monomers and the heterodimer unfolded in 4 M urea. The observed rates were virtually identical above 0.4 M urea, exhibiting a log-linear relationship on the final denaturant concentration. Below approximately 0.4 M urea (concentrations inaccessible from the 4-M urea unfolded state), a rollover in the rates was observed; this suggests that a component of the I(2) ensemble contains non-native structure that rearranges/isomerizes to a more native-like species. The contribution of helix propensity to the stability of the I(2) ensemble was assessed with a set of H2A-H2B mutants containing Ala and Gly replacements at nine sites, focusing mainly on the long, central alpha2 helix. Equilibrium and kinetic folding/unfolding data were collected to determine the effects of the mutations on the stability of I(2) and the transition state between I(2) and N(2). This limited mutational study indicated that residues in the alpha2 helices of H2A and H2B as well as alpha1 of H2B and both the C-terminus of alpha3 and the short alphaC helix of H2A contribute to the stability of the I(2) burst-phase species. Interestingly, at least eight of the nine targeted residues stabilize I(2) by interactions that are non-native to some extent. Given that destabilizing I(2) and these non-native interactions does not accelerate folding, it is concluded that the native and non-native structures present in the I(2) ensemble enable efficient folding of H2A-H2B.

post to: CiteULike

Structure-function analysis of the short splicing variant carboxypeptidase encoded by Drosophila melanogaster silver.

Publication Date: 2010 Aug 20 PMID: 20600119
Authors: Tanco, S. - Arolas, J. L. - Guevara, T. - Lorenzo, J. - Aviles, F. X. - Gomis-Ruth, F. X.
Journal: J Mol Biol

Drosophila melanogaster silver gene is the ortholog of the coding gene of mammalian carboxypeptidase D (CPD). The silver gene gives rise to eight different splicing variants of differing length that can contain up to three homologous repeats. Among the protein variants encoded, the short form 1B alias DmCPD1Bs (D. melanogaster CPD variant 1B short) is necessary and sufficient for viability of the fruit fly. It has one single repeat, it is active against standard peptide substrates, and it is localized to the secretory pathway. In this work, the enzyme was found as a monomer in solution and as a homodimer in the crystal structure, which features a protomer with an N-terminal 311-residue catalytic domain of alpha/beta-hydrolase fold and a C-terminal 84-residue all-beta transthyretin-like domain. Overall, DmCPD1Bs conforms to the structure of N/E-type funnelins/M14B metallopeptidases, but it has two unique structural elements potentially involved in regulation of its activity: (i) two contiguous surface cysteines that may become palmitoylated and target the enzyme to membranes, thus providing control through localization, and (ii) a surface hot spot targetable by peptidases that would provide a regulatory mechanism through proteolytic inactivation. Given that the fruit fly possesses orthologs of only two out of the five proteolytically competent N/E-type funnelins found in higher vertebrates, DmCPD1Bs may represent a functional analog of at least one of the missing mammalian CPs.

post to: CiteULike

The NusA N-terminal domain is necessary and sufficient for enhancement of transcriptional pausing via interaction with the RNA exit channel of RNA polymerase.

Publication Date: 2010 Sep 3 PMID: 20600118
Authors: Ha, K. S. - Toulokhonov, I. - Vassylyev, D. G. - Landick, R.
Journal: J Mol Biol

NusA is a core, multidomain regulator of transcript elongation in bacteria and archaea. Bacterial NusA interacts with elongating complexes and the nascent RNA transcript in ways that stimulate pausing and termination but that can be switched to antipausing and antitermination by other accessory proteins. This regulatory complexity of NusA likely depends on its multidomain structure, but it remains unclear which NusA domains possess which regulatory activity and how they interact with elongating RNA polymerase. We used a series of truncated NusA proteins to measure the effect of the NusA domains on transcriptional pausing and termination. We find that the N-terminal domain (NTD) of NusA is necessary and sufficient for enhancement of transcriptional pausing and that the other NusA domains contribute to NusA binding to elongating complexes. Stimulation of intrinsic termination requires higher concentrations of NusA and involves both the NTD and other NusA domains. Using a tethered chemical protease in addition to protein-RNA cross-linking, we show that the NusA NTD contacts the RNA exit channel of RNA polymerase. Finally, we report evidence that the NusA NTD recognizes duplex RNA in the RNA exit channel.

post to: CiteULike

Equivalent mutations in the eight subunits of the chaperonin CCT produce dramatically different cellular and gene expression phenotypes.

Publication Date: 2010 Aug 20 PMID: 20600117
Authors: Amit, M. - Weisberg, S. J. - Nadler-Holly, M. - McCormack, E. A. - Feldmesser, E. - Kaganovich, D. - Willison, K. R. - Horovitz, A.
Journal: J Mol Biol

The eukaryotic cytoplasmic chaperonin-containing TCP-1 (CCT) is a complex formed by two back-to-back stacked hetero-octameric rings that assists the folding of actins, tubulins, and other proteins in an ATP-dependent manner. Here, we tested the significance of the hetero-oligomeric nature of CCT in its function by introducing, in each of the eight subunits in turn, an identical mutation at a position that is conserved in all the subunits and is involved in ATP hydrolysis, in order to establish the extent of 'individuality' of the various subunits. Our results show that these identical mutations lead to dramatically different phenotypes. For example, Saccharomyces cerevisiae yeast cells with the mutation in subunit CCT2 display heat sensitivity and cold sensitivity for growth, have an excess of actin patches, and are the only strain here generated that is pseudo-diploid. By contrast, cells with the mutation in subunit CCT7 are the only ones to accumulate juxtanuclear protein aggregates that may reflect an impaired stress response in this strain. System-level analysis of the strains using RNA microarrays reveals connections between CCT and several cellular networks, including ribosome biogenesis and TOR2, that help to explain the phenotypic variability observed.

post to: CiteULike

Structure and function of P19, a high-affinity iron transporter of the human pathogen Campylobacter jejuni.

Publication Date: 2010 Aug 27 PMID: 20600116
Authors: Chan, A. C. - Doukov, T. I. - Scofield, M. - Tom-Yew, S. A. - Ramin, A. B. - Mackichan, J. K. - Gaynor, E. C. - Murphy, M. E.
Journal: J Mol Biol

Campylobacter jejuni, a major cause of acute bacterial diarrhea in humans, expresses numerous proteins to import diverse forms of essential iron. The expression of p19 and an adjacent iron transporter homologue (ftr1) is strongly induced upon iron limitation, suggesting a function in iron acquisition. Here, we show that the loss of P19 alone is detrimental to growth on iron-restricted media. Furthermore, metal binding analysis demonstrates that recombinant P19 has distinct copper and iron binding sites. Crystal structures of P19 have been solved to 1.41 A resolution, revealing an immunoglobulin-like fold. A P19 homodimer in which both monomers contribute ligands to two equivalent copper sites located adjacent to methionine-rich patches is observed. Copper coordination occurs via three histidine residues (His42, His95, and His132) and Met88. A solvent channel lined with conserved acidic residues leads to the copper site. Soaking crystals with a solution of manganese as iron analog reveals a second metal binding site in this solvent channel (metal-metal distance, 7.7 A). Glu44 lies between the metal sites and displays multiple conformations in the crystal structures, suggesting a role in regulating metal-metal interaction. Dimerization is shown to be metal dependent in vitro and is detected in vivo by cross-linking.

MeSH Categories: Bacterial Proteins/*chemistry/metabolism, Binding Sites, Campylobacter jejuni/*metabolism/pathogenicity, Copper/chemistry/metabolism, Crystallography, X-Ray, Humans, Iron/*metabolism, Ligands, Manganese/metabolism, Membrane Transport Proteins/*chemistry/metabolism, Models, Molecular, Molecular Sequence Data, Structure-Activity Relationship

post to: CiteULike

Disulfide bond stabilization of the hexameric capsomer of human immunodeficiency virus.

Publication Date: 2010 Sep 3 PMID: 20600115
Authors: Pornillos, O. - Ganser-Pornillos, B. K. - Banumathi, S. - Hua, Y. - Yeager, M.
Journal: J Mol Biol

The human immunodeficiency virus type 1 capsid is modeled as a fullerene cone that is composed of approximately 250 hexamers and 12 pentamers of the viral CA protein. Structures of CA hexamers have been difficult to obtain because the hexamer-stabilizing interactions are inherently weak, and CA tends to spontaneously assemble into capsid-like particles. Here, we describe a two-step biochemical strategy to obtain soluble CA hexamers for crystallization. First, the hexamer was stabilized by engineering disulfide cross-links (either A14C/E45C or A42C/T54C) between the N-terminal domains of adjacent subunits. Second, the cross-linked hexamers were prevented from polymerizing further into hyperstable capsid-like structures by mutations (W184A and M185A) that interfered with dimeric association between the C-terminal domains that link adjacent hexamers. The structures of two different cross-linked CA hexamers were nearly identical, and we combined the non-mutated portions of the structures to generate an atomic resolution model for the native hexamer. This hybrid approach for structure determination should be applicable to other viral capsomers and protein-protein complexes in general.

post to: CiteULike

A G-C-rich palindromic structural motif and a stretch of single-stranded purines are required for optimal packaging of Mason-Pfizer monkey virus (MPMV) genomic RNA.

Publication Date: 2010 Sep 3 PMID: 20600114
Authors: Jaballah, S. A. - Aktar, S. J. - Ali, J. - Phillip, P. S. - Al Dhaheri, N. S. - Jabeen, A. - Rizvi, T. A.
Journal: J Mol Biol

During retroviral RNA packaging, two copies of genomic RNA are preferentially packaged into the budding virus particles whereas the spliced viral RNAs and the cellular RNAs are excluded during this process. Specificity towards retroviral RNA packaging is dependent upon sequences at the 5' end of the viral genome, which at times extend into Gag sequences. It has earlier been suggested that the Mason-Pfizer monkey virus (MPMV) contains packaging sequences within the 5' untranslated region (UTR) and Gag. These studies have also suggested that the packaging determinants of MPMV that lie in the UTR are bipartite and are divided into two regions both upstream and downstream of the major splice donor. However, the precise boundaries of these discontinuous regions within the UTR and the role of the intervening sequences between these dipartite sequences towards MPMV packaging have not been investigated. Employing a combination of genetic and structural prediction analyses, we have shown that region "A", immediately downstream of the primer binding site, is composed of 50 nt, whereas region "B" is composed of the last 23 nt of UTR, and the intervening 55 nt between these two discontinuous regions do not contribute towards MPMV RNA packaging. In addition, we have identified a 14-nt G-C-rich palindromic sequence (with 100% autocomplementarity) within region A that has been predicted to fold into a structural motif and is essential for optimal MPMV RNA packaging. Furthermore, we have also identified a stretch of single-stranded purines (ssPurines) within the UTR and 8 nt of these ssPurines are duplicated in region B. The native ssPurines or its repeat in region B when predicted to refold as ssPurines has been shown to be essential for RNA packaging, possibly functioning as a potential nucleocapsid binding site. Findings from this study should enhance our understanding of the steps involved in MPMV replication including RNA encapsidation process.

post to: CiteULike

Structural characterization of the rod cGMP phosphodiesterase 6.

Publication Date: 2010 Aug 20 PMID: 20600113
Authors: Goc, A. - Chami, M. - Lodowski, D. T. - Bosshart, P. - Moiseenkova-Bell, V. - Baehr, W. - Engel, A. - Palczewski, K.
Journal: J Mol Biol

Rod cGMP phosphodiesterase 6 (PDE6) is a key enzyme of the phototransduction cascade, consisting of PDE6alpha, PDE6beta, and two regulatory PDE6gamma subunits. PDE6 is membrane associated through isoprenyl membrane anchors attached to the C-termini of PDE6alpha and PDE6beta and can form a complex with prenyl-binding protein delta (PrBP/delta), an isoprenyl-binding protein that is highly expressed in photoreceptors. The stoichiometry of PDE6-PrBP/delta binding and the mechanism by which the PDE6-PrBP/delta complex assembles have not been fully characterized, and the location of regulatory PDE6gamma subunits within the protein assembly has not been elucidated. To clarify these questions, we have developed a rapid purification method for PDE6-PrBP/delta from bovine rod outer segments utilizing recombinant PrBP/delta. Transmission electron microscopy of negatively stained samples revealed the location of PrBP/delta and, thus, where the carboxyl-termini of PDE6alpha and PDE6beta must be located. The three-dimensional structure of the PDE6alphabetagamma complex was determined up to 18 A resolution from single-particle projections and was interpreted by model building to identify the probable location of isoprenylation, PDE6gamma subunits, and catalytic sites.

post to: CiteULike

Structure of the catalytic a(0)a fragment of the protein disulfide isomerase ERp72.

Publication Date: 2010 Aug 27 PMID: 20600112
Authors: Kozlov, G. - Azeroual, S. - Rosenauer, A. - Maattanen, P. - Denisov, A. Y. - Thomas, D. Y. - Gehring, K.
Journal: J Mol Biol

Protein disulfide isomerases (PDIs) are responsible for catalyzing the proper oxidation and isomerization of disulfide bonds of newly synthesized proteins in the endoplasmic reticulum (ER). The ER contains many different PDI-like proteins. Some, such as PDI, are general enzymes that directly recognize misfolded proteins while others, such as ERp57 and ERp72, have more specialized roles. Here, we report the high-resolution X-ray crystal structure of the N-terminal portion of ERp72 (also known as CaBP2 or PDI A4), which contains two a(0)a catalytic thioredoxin-like domains. The structure shows that the a(0) domain contains an additional N-terminal beta-strand and a different conformation of the beta5-alpha4 loop relative to other thioredoxin-like domains. The structure of the a domain reveals that a conserved arginine residue inserts into the hydrophobic core and makes a salt bridge with a conserved glutamate residue in the vicinity of the catalytic site. A structural model of full-length ERp72 shows that all three catalytic sites roughly face each other and positions the adjacent hydrophobic patches that are likely involved in protein substrate binding.

MeSH Categories: Binding Sites, Catalytic Domain, Disulfides, Endoplasmic Reticulum/metabolism, Hydrophobicity, Membrane Glycoproteins/*chemistry/metabolism, Protein Disulfide-Isomerases/*chemistry/metabolism, Protein Folding, Thioredoxins/chemistry/metabolism

post to: CiteULike

Crystal structure of Methanocaldococcus jannaschii Trm4 complexed with sinefungin.

Publication Date: 2010 Aug 20 PMID: 20600111
Authors: Kuratani, M. - Hirano, M. - Goto-Ito, S. - Itoh, Y. - Hikida, Y. - Nishimoto, M. - Sekine, S. - Bessho, Y. - Ito, T. - Grosjean, H. - Yokoyama, S.
Journal: J Mol Biol

tRNA:m(5)C methyltransferase Trm4 generates the modified nucleotide 5-methylcytidine in archaeal and eukaryotic tRNA molecules, using S-adenosyl-l-methionine (AdoMet) as methyl donor. Most archaea and eukaryotes possess several Trm4 homologs, including those related to diseases, while the archaeon Methanocaldococcus jannaschii has only one gene encoding a Trm4 homolog, MJ0026. The recombinant MJ0026 protein catalyzed AdoMet-dependent methyltransferase activity on tRNA in vitro and was shown to be the M. jannaschii Trm4. We determined the crystal structures of the substrate-free M. jannaschii Trm4 and its complex with sinefungin at 1.27 A and 2.3 A resolutions, respectively. This AdoMet analog is bound in a negatively charged pocket near helix alpha8. This helix can adopt two different conformations, thereby controlling the entry of AdoMet into the active site. Adjacent to the sinefungin-bound pocket, highly conserved residues form a large, positively charged surface, which seems to be suitable for tRNA binding. The structure explains the roles of several conserved residues that were reportedly involved in the enzymatic activity or stability of Trm4p from the yeast Saccharomyces cerevisiae. We also discuss previous genetic and biochemical data on human NSUN2/hTrm4/Misu and archaeal PAB1947 methyltransferase, based on the structure of M. jannaschii Trm4.

post to: CiteULike

Protein S20 binds two 16S rRNA sites as assembly is initiated.

Publication Date: 2010 Aug 20 PMID: 20600110
Authors: Hedrick, E. G. - Hill, W. E.
Journal: J Mol Biol

Ribosomal protein S20 is a primary binding protein that bridges the 5' domain and the 3' minor domain of the 16S ribosomal RNA (rRNA) in the 30S ribosomal subunit. Using time-dependent dimethyl sulfate modification, we have determined that as it is bound to 16S rRNA, protein S20 causes rapid protection of bases A246, A274, A279, and A282 in the stem region of helix 11 in the 5' domain and moderately fast modifications of helix 44 bases A1433 and A1434 in the 3' minor domain. At a later time, enhancements occur with bases A181and A190 in helix 9, bases A325 and A327 in helix 13, and base C264 at the distal end of helix 11 in the 5' domain of 16S rRNA. The modifications that occur in the stem region of helix 11 are distant from the binding site of protein S20, as determined from the crystal structure. Simultaneous addition of protein S17 with S20 to the complex significantly alters the modifications caused by protein S20 in the stem region of helix 11 but does not alter the remaining modifications. Our results indicate that protein S20 is binding to at least two alternate 16S rRNA sites during the early assembly process.

post to: CiteULike

High-resolution orientation and depth of insertion of the voltage-sensing S4 helix of a potassium channel in lipid bilayers.

Publication Date: 2010 Aug 27 PMID: 20600109
Authors: Doherty, T. - Su, Y. - Hong, M.
Journal: J Mol Biol

The opening and closing of voltage-gated potassium (Kv) channels are controlled by several conserved Arg residues in the S4 helix of the voltage-sensing domain. The interaction of these positively charged Arg residues with the lipid membrane has been of intense interest for understanding how membrane proteins fold to allow charged residues to insert into lipid bilayers against free-energy barriers. Using solid-state NMR, we have now determined the orientation and insertion depth of the S4 peptide of the KvAP channel in lipid bilayers. Two-dimensional (15)N correlation experiments of macroscopically oriented S4 peptide in phospholipid bilayers revealed a tilt angle of 40 degrees and two possible rotation angles differing by 180 degrees around the helix axis. Remarkably, the tilt angle and one of the two rotation angles are identical to those of the S4 helix in the intact voltage-sensing domain, suggesting that interactions between the S4 segment and other helices of the voltage-sensing domain are not essential for the membrane topology of the S4 helix. (13)C-(31)P distances between the S4 backbone and the lipid (31)P indicate a approximately 9 A local thinning and 2 A average thinning of the DMPC (1,2-dimyristoyl-sn-glycero-3-phosphochloline)/DMPG (1,2-dimyristoyl-sn-glycero-3-phosphatidylglycerol) bilayer, consistent with neutron diffraction data. Moreover, a short distance of 4.6 A from the guanidinium C(zeta) of the second Arg to (31)P indicates the existence of guanidinium phosphate hydrogen bonding and salt bridges. These data suggest that the structure of the Kv gating helix is mainly determined by protein-lipid interactions instead of interhelical protein-protein interactions, and the S4 amino acid sequence encodes sufficient information for the membrane topology of this crucial gating helix.

MeSH Categories: Arginine/chemistry/metabolism, Dimyristoylphosphatidylcholine/chemistry, Ion Channel Gating, Lipid Bilayers/*chemistry, Magnetic Resonance Spectroscopy, Potassium Channels/*chemistry, Protein Structure, Secondary

post to: CiteULike

Helical filaments of human Dmc1 protein on single-stranded DNA: a cautionary tale.

Publication Date: 2010 Aug 20 PMID: 20600108
Authors: Yu, X. - Egelman, E. H.
Journal: J Mol Biol

Proteins in the RecA/Rad51/RadA family form nucleoprotein filaments on DNA that catalyze a strand exchange reaction as part of homologous genetic recombination. Because of the centrality of this system to many aspects of DNA repair, the generation of genetic diversity, and cancer when this system fails or is not properly regulated, these filaments have been the object of many biochemical and biophysical studies. A recent paper has argued that the human Dmc1 protein, a meiotic homolog of bacterial RecA and human Rad51, forms filaments on single-stranded DNA with approximately 9 subunits per turn in contrast to the filaments formed on double-stranded DNA with approximately 6.4 subunits per turn and that the stoichiometry of DNA binding is different between these two filaments. We show using scanning transmission electron microscopy that the Dmc1 filament formed on single-stranded DNA has a mass per unit length expected from approximately 6.5 subunits per turn. More generally, we show how ambiguities in helical symmetry determination can generate incorrect solutions and why one sometimes must use other techniques, such as biochemistry, metal shadowing, or scanning transmission electron microscopy, to resolve these ambiguities. While three-dimensional reconstruction of helical filaments from EM images is a powerful tool, the intrinsic ambiguities that may be present with limited resolution are not sufficiently appreciated.

post to: CiteULike

Action of the chaperonin GroEL/ES on a non-native substrate observed with single-molecule FRET.

Publication Date: 2010 Aug 27 PMID: 20600107
Authors: Kim, S. Y. - Miller, E. J. - Frydman, J. - Moerner, W. E.
Journal: J Mol Biol

The double ring-shaped chaperonin GroEL binds a wide range of non-native polypeptides within its central cavity and, together with its cofactor GroES, assists their folding in an ATP-dependent manner. The conformational cycle of GroEL/ES has been studied extensively but little is known about how the environment in the central cavity affects substrate conformation. Here, we use the von Hippel-Lindau tumor suppressor protein VHL as a model substrate for studying the action of the GroEL/ES system on a bound polypeptide. Fluorescent labeling of pairs of sites on VHL for fluorescence (Forster) resonant energy transfer (FRET) allows VHL to be used to explore how GroEL binding and GroEL/ES/nucleotide binding affect the substrate conformation. On average, upon binding to GroEL, all pairs of labeling sites experience compaction relative to the unfolded protein while single-molecule FRET distributions show significant heterogeneity. Upon addition of GroES and ATP to close the GroEL cavity, on average further FRET increases occur between the two hydrophobic regions of VHL, accompanied by FRET decreases between the N- and C-termini. This suggests that ATP- and GroES-induced confinement within the GroEL cavity remodels bound polypeptides by causing expansion (or racking) of some regions and compaction of others, most notably, the hydrophobic core. However, single-molecule observations of the specific FRET changes for individual proteins at the moment of ATP/GroES addition reveal that a large fraction of the population shows the opposite behavior; that is, FRET decreases between the hydrophobic regions and FRET increases for the N- and C-termini. Our time-resolved single-molecule analysis reveals the underlying heterogeneity of the action of GroES/EL on a bound polypeptide substrate, which might arise from the random nature of the specific binding to the various identical subunits of GroEL, and might help explain why multiple rounds of binding and hydrolysis are required for some chaperonin substrates.

MeSH Categories: Binding Sites, Chaperonin 10/*chemistry/metabolism, Chaperonin 60/*chemistry/metabolism, Energy Transfer, Fluorescence Resonance Energy Transfer/*methods, Hydrolysis, Models, Molecular, Protein Conformation, Substrate Specificity

post to: CiteULike

Positive autoregulation shapes response timing and intensity in two-component signal transduction systems.

Publication Date: 2010 Aug 27 PMID: 20600106
Authors: Mitrophanov, A. Y. - Hadley, T. J. - Groisman, E. A.
Journal: J Mol Biol

Positive feedback loops are regulatory elements that can modulate expression output, kinetics and noise in genetic circuits. Transcriptional regulators participating in such loops are often expressed from two promoters, one constitutive and one autoregulated. Here, we investigate the interplay of promoter strengths and the intensity of the stimulus activating the transcriptional regulator in defining the output of a positively autoregulated genetic circuit. Using a mathematical model of two-component regulatory systems, which are present in all domains of life, we establish that positive feedback strongly affects the steady-state output levels at both low and high levels of stimulus if the constitutive promoter of the regulator is weak. By contrast, the effect of positive feedback is negligible when the constitutive promoter is sufficiently strong, unless the stimulus intensity is very high. Furthermore, we determine that positive feedback can affect both transient and steady state output levels even in the simplest genetic regulatory systems. We tested our modeling predictions by abolishing the positive feedback loop in the two-component regulatory system PhoP/PhoQ of Salmonella enterica, which resulted in diminished induction of PhoP-activated genes.

MeSH Categories: Bacterial Proteins/*genetics/metabolism, *Gene Expression Regulation, Bacterial, Homeostasis/genetics, Promoter Regions, Genetic, Salmonella enterica/genetics/metabolism, Signal Transduction/*genetics, Transcription, Genetic

post to: CiteULike

Crystal structures of inhibitor complexes of human T-cell leukemia virus (HTLV-1) protease.

Publication Date: 2010 Aug 27 PMID: 20600105
Authors: Satoh, T. - Li, M. - Nguyen, J. T. - Kiso, Y. - Gustchina, A. - Wlodawer, A.
Journal: J Mol Biol

Human T-cell leukemia virus type 1 (HTLV-1) is a retrovirus associated with several serious diseases, such as adult T-cell leukemia and tropical spastic paraparesis/myelopathy. For a number of years, the protease (PR) encoded by HTLV-1 has been a target for designing antiviral drugs, but that effort was hampered by limited available structural information. We report a high-resolution crystal structure of HTLV-1 PR complexed with a statine-containing inhibitor, a significant improvement over the previously available moderate-resolution structure. We also report crystal structures of the complexes of HTLV-1 PR with five different inhibitors that are more compact and more potent. A detailed study of structure-activity relationships was performed to interpret in detail the influence of the polar and hydrophobic interactions between the inhibitors and the protease.

MeSH Categories: Aspartic Acid Endopeptidases/*chemistry/metabolism, Crystallography, X-Ray, Human T-lymphotropic virus 1/*enzymology, Humans, Hydrophobicity, Protease Inhibitors/*chemistry/metabolism, Structure-Activity Relationship

post to: CiteULike

Biophysical characterisation of fibulin-5 proteins associated with disease.

Publication Date: 2010 Aug 27 PMID: 20599547
Authors: Schneider, R. - Jensen, S. A. - Whiteman, P. - McCullagh, J. S. - Redfield, C. - Handford, P. A.
Journal: J Mol Biol

FBLN5 encodes fibulin-5, an extracellular matrix calcium-binding glycoprotein that is essential for elastic fibre formation. FBLN5 mutations are associated with two distinct human diseases, age-related macular degeneration (AMD) and cutis laxa (CL), but the biochemical basis for the pathogenic effects of these mutations is poorly understood. Two missense mutations found in AMD patients (I169T and G267S) and two missense mutations found in CL patients (G202R and S227P) were analysed in a native-like context in recombinant fibulin-5 fragments. Limited proteolysis, NMR spectroscopy and chromophoric calcium chelation experiments showed that the G267S and S227P substitutions cause long-range structural effects consistent with protein misfolding. Cellular studies using fibroblast cells further demonstrated that these recombinant forms of mutant fibulin-5 were not present in the extracellular medium, consistent with retention. In contrast, no significant effects of I169T and G202R substitutions on protein fold and secretion were identified. These data establish protein misfolding as a causative basis for the effects of G267S and S227P substitutions in AMD and CL, respectively, and raise the possibility that the I169T and G202R substitutions may be polymorphisms or may increase susceptibility to disease.

MeSH Categories: Cutis Laxa/genetics/metabolism, Extracellular Matrix Proteins/*chemistry/genetics/metabolism, Genetic Predisposition to Disease, Humans, Macular Degeneration/genetics/metabolism, Magnetic Resonance Spectroscopy, Models, Molecular, Mutation, Missense, Protein Conformation, Protein Folding

post to: CiteULike

Structural and dynamic determinants of ligand binding and regulation of cyclin-dependent kinase 5 by pathological activator p25 and inhibitory peptide CIP.

Publication Date: 2010 Aug 20 PMID: 20599546
Authors: Cardone, A. - Hassan, S. A. - Albers, R. W. - Sriram, R. D. - Pant, H. C.
Journal: J Mol Biol

The crystal structure of the cdk5/p25 complex has provided information on possible molecular mechanisms of the ligand binding, specificity, and regulation of the kinase. Comparative molecular dynamics simulations are reported here for physiological conditions. This study provides new insight on the mechanisms that modulate such processes, which may be exploited to control pathological activation by p25. The structural changes observed in the kinase are stabilized by a network of interactions involving highly conserved residues within the cyclin-dependent kinase (cdk) family. Collective motions of the proteins (cdk5, p25, and CIP) and their complexes are identified by principal component analysis, revealing two conformational states of the activation loop upon p25 complexation, which are absent in the uncomplexed kinase and not apparent from the crystal. Simulations of the uncomplexed inhibitor CIP show structural rearrangements and increased flexibility of the interfacial loop containing the critical residue E240, which becomes fully hydrated and available for interactions with one of several positively charged residues in the kinase. These changes provide a rationale for the observed high affinity and enhanced inhibitory action of CIP when compared to either p25 or the physiological activators of cdk5.

post to: CiteULike

The CcmC:heme:CcmE complex in heme trafficking and cytochrome c biosynthesis.

Publication Date: 2010 Aug 20 PMID: 20599545
Authors: Richard-Fogal, C. - Kranz, R. G.
Journal: J Mol Biol

A superfamily of integral membrane proteins is characterized by a conserved tryptophan-rich region (called the WWD domain) in an external loop at the inner membrane surface. The three major members of this family (CcmC, CcmF, and CcsBA) are each involved in cytochrome c biosynthesis, yet the function of the WWD domain is unknown. It has been hypothesized that the WWD domain binds heme to present it to an acceptor protein (apoCcmE for CcmC or apocytochrome c for CcmF and CcsBA) such that the heme vinyl group(s) covalently attaches to the acceptors. Alternative proposals suggest that the WWD domain interacts directly with the acceptor protein (e.g., apoCcmE for CcmC). Here, it is shown that CcmC is only trapped with heme when its cognate acceptor protein CcmE is present. It is demonstrated that CcmE only interacts stably with CcmC when heme is present; thus, specific residues in each protein provide sites of interaction with heme to form this very stable complex. For the first time, evidence that the external WWD domain of CcmC interacts directly with heme is presented. Single and multiple substitutions of completely conserved residues in the WWD domain of CcmC alter the spectral properties of heme in the stable CcmC:heme:CcmE complexes. Moreover, some mutations reduce the binding of heme up to 100%. It is likely that endogenously synthesized heme enters the external WWD domain of CcmC either via a channel within this six-transmembrane-spanning protein or from the membrane. The data suggest that a specific heme channel (i.e., heme binding site within membrane spanning helices) is not present in CcmC, in contrast to the CcsBA protein. We discuss the likelihood that it is not important to protect the heme via trafficking in CcmC whereas it is critical in CcsBA.

post to: CiteULike

Protein-protein interactions between sigma(70) region 4 of RNA polymerase and Escherichia coli SoxS, a transcription activator that functions by the prerecruitment mechanism: evidence for "off-DNA" and "on-DNA" interactions.

Publication Date: 2010 Aug 6 PMID: 20595001
Authors: Zafar, M. A. - Shah, I. M. - Wolf, R. E. Jr
Journal: J Mol Biol

According to the prerecruitment hypothesis, Escherichia coli SoxS activates the transcription of the genes of the SoxRS regulon by forming binary complexes with RNA polymerase (RNAP) that scan the chromosome for class I and class II SoxS-dependent promoters. We showed previously that the alpha subunit's C-terminal domain plays a role in activating both classes of promoter by making protein-protein contacts with SoxS; some of these contacts are made in solution in the absence of promoter DNA, a critical prediction of the prerecruitment hypothesis. Here, we identified seven single-alanine substitutions of the region 4 of sigma(70) (sigma(70) R4) of RNAP that reduce SoxS activation of class II promoters. With genetic epistasis tests between these sigma(70) R4 mutants and positive control mutants of SoxS, we identified 10 pairs of amino acids that interact with each other in E. coli. Using the yeast two-hybrid system and affinity immobilization assays, we showed that SoxS and sigma(70) R4 can interact in solution (i.e., "off-DNA"). The interaction requires amino acids of the class I/II (but not the class II) positive control surface of SoxS, and five amino acids of sigma(70) R4 that reduce activation in E. coli also reduce the SoxS-sigma(70) R4 interaction in yeast. One of the epistatic interactions that occur in E. coli also occurs in the yeast two-hybrid system (i.e., off-DNA). Importantly, we infer that the five epistatic interactions occurring in E. coli that require an amino acid of the class II surface occur "on-DNA" at class II promoters. Finding that SoxS contacts sigma(70) R4 both off-DNA and on-DNA is consistent with the prerecruitment hypothesis. Moreover, SoxS is now the first example of an E. coli transcriptional activator that uses a single positive control surface to make specific protein-protein contacts with two different subunits of RNAP.

MeSH Categories: Alanine/genetics, Amino Acid Substitution/genetics/physiology, Arginine/genetics, DNA, Bacterial/*metabolism/physiology, DNA-Directed RNA Polymerases/chemistry/genetics/*metabolism, Epistasis, Genetic, Escherichia coli/genetics/metabolism, Escherichia coli Proteins/chemistry/genetics/*metabolism, *Gene Expression Regulation, Bacterial, Organisms, Genetically Modified, Promoter Regions, Genetic, Protein Binding/genetics/physiology, Protein Interaction Domains and Motifs/genetics, Protein Interaction Mapping, Sigma Factor/chemistry/genetics/*metabolism, Trans-Activators/*metabolism, Two-Hybrid System Techniques

post to: CiteULike

A critical assessment of putative gatekeeper interactions in the villin headpiece helical subdomain.

Publication Date: 2010 Aug 13 PMID: 20570680
Authors: Xiao, S. - Raleigh, D. P.
Journal: J Mol Biol

The helical subdomain of the villin headpiece (HP36) is one of the smallest naturally occurring proteins that folds cooperatively. Its small size, rapid folding, and simple three-helix topology have made it an extraordinary popular model system for computational, theoretical, and experimental studies of protein folding. Aromatic-proline interactions involving Trp64 and Pro62 have been proposed to play a critical role in specifying the subdomain fold by acting as gatekeeper residues. Note that the numbering corresponds to full-length headpiece. Mutation of Pro62 has been shown to lead to a protein that does not fold, but this may arise for two different reasons: The residue may make interactions that are critical for the specificity of the fold or the mutation may simply destabilize the domain. In the first case, the protein cannot fold, while in the second, the small fraction of molecules that do fold adopt the correct structure. The modest stability of the wild type prevents a critical analysis of these interactions because even moderately destabilizing mutations lead to a very small folded state population. Using a hyperstable variant of HP36, denoted DM HP36, as our new wild type, we characterized a set of mutants designed to assess the role of the putative gatekeeper interactions. Four single mutants, DM Pro62Ala, DM Trp64Leu, DM Trp64Lys, and DM Trp64Ala, and a double mutant, DM Pro62Ala Trp64Leu, were prepared. All mutants are less stable than DM HP36, but all are well folded as judged by CD and (1)H NMR. All of the mutants display sigmoidal thermal unfolding and urea-induced unfolding curves. Double-mutant cycle analysis shows that the interactions between Pro62 and Trp64 are weak but favorable. Interactions involving Pro62 and proline-aromatic interactions are, thus, not required for specifying the subdomain fold. The implications for the design and thermodynamics of miniature proteins are discussed.

MeSH Categories: Amino Acid Sequence, Amino Acid Substitution, Circular Dichroism, Models, Molecular, Molecular Dynamics Simulation, Molecular Sequence Data, Mutagenesis, Site-Directed, Neurofilament Proteins/*chemistry/genetics, Nuclear Magnetic Resonance, Biomolecular, Peptide Fragments/*chemistry/genetics, Protein Folding, Protein Interaction Domains and Motifs, Protein Stability, Recombinant Proteins/chemistry/genetics, Thermodynamics

post to: CiteULike

Correlation between the OmpG secondary structure and its pH-dependent alterations monitored by FTIR.

Publication Date: 2010 Aug 6 PMID: 20561532
Authors: Korkmaz-Ozkan, F. - Koster, S. - Kuhlbrandt, W. - Mantele, W. - Yildiz, O.
Journal: J Mol Biol

The channel activity of the outer-membrane protein G (OmpG) from Escherichia coli is pH-dependent. To investigate the role of the histidine pair His231/His261 in triggering channel opening and closing, we mutated both histidines to alanines and cysteines. Fourier transform infrared spectra revealed that the OmpG mutants stay-independent of pH-in an open conformation. Temperature ramp experiments indicate that the mutants are as stable as the open state of wild-type OmpG. The X-ray structure of the alanine-substituted OmpG mutant obtained at pH 6.5 confirms the constitutively open conformation. Compared to previous structures of the wild-type protein in the open and closed conformation, the mutant structure shows a difference in the extracellular loop L6 connecting beta-strands S12 and S13. A deletion of amino acids 220-228, which are thought to block the channel at low pH in wild-type OmpG, indicates conformational changes, which might be triggered by His231/His261.

MeSH Categories: Bacterial Outer Membrane Proteins/*chemistry/drug effects/genetics, Crystallography, X-Ray, Detergents/pharmacology, Escherichia coli Proteins/*chemistry/drug effects/genetics, Hydrogen-Ion Concentration, Ion Channels/chemistry/genetics/metabolism, Lipids/pharmacology, Models, Molecular, Organisms, Genetically Modified, Porins/*chemistry/drug effects/genetics, Protein Folding, Protein Stability/drug effects, Protein Structure, Secondary/drug effects/genetics, Spectroscopy, Fourier Transform Infrared, Thermodynamics

post to: CiteULike

Structural basis for Par-4 recognition by the SPRY domain- and SOCS box-containing proteins SPSB1, SPSB2, and SPSB4.

Publication Date: 2010 Aug 20 PMID: 20561531
Authors: Filippakopoulos, P. - Low, A. - Sharpe, T. D. - Uppenberg, J. - Yao, S. - Kuang, Z. - Savitsky, P. - Lewis, R. S. - Nicholson, S. E. - Norton, R. S. - Bullock, A. N.
Journal: J Mol Biol

The mammalian SPRY domain- and SOCS box-containing proteins, SPSB1 to SPSB4, belong to the SOCS box family of E3 ubiquitin ligases. Substrate recognition sites for the SPRY domain are identified only for human Par-4 (ELNNNL) and for the Drosophila orthologue GUSTAVUS binding to the DEAD-box RNA helicase VASA (DINNNN). To further investigate this consensus motif, we determined the crystal structures of SPSB1, SPSB2, and SPSB4, as well as their binding modes and affinities for both Par-4 and VASA. Mutation of each of the three Asn residues in Par-4 abrogated binding to all three SPSB proteins, while changing EL to DI enhanced binding. By comparison to SPSB1 and SPSB4, the more divergent protein SPSB2 showed only weak binding to Par-4 and was hypersensitive to DI substitution. Par-4((59-77)) binding perturbed NMR resonances from a number of SPSB2 residues flanking the ELNNN binding site, including loop D, which binds the EL/DI sequence. Although interactions with the consensus peptide motif were conserved in all structures, flanking sites in SPSB2 were identified as sites of structural change. These structural changes limit high-affinity interactions for SPSB2 to aspartate-containing sequences, whereas SPSB1 and SPSB4 bind strongly to both Par-4 and VASA peptides.

post to: CiteULike

Structure and specificity of a binary tandem domain F-lectin from striped bass (Morone saxatilis).

Publication Date: 2010 Aug 13 PMID: 20561530
Authors: Bianchet, M. A. - Odom, E. W. - Vasta, G. R. - Amzel, L. M.
Journal: J Mol Biol

The plasma of the striped bass Morone saxatilis contains a fucose-specific lectin (MsaFBP32) that consists of two F-type carbohydrate recognition domains (CRDs) in tandem. The crystal structure of the complex of MsaFBP32 with l-fucose reported here shows a cylindrical 81-A-long and 60-A-wide trimer divided into two globular halves: one containing N-terminal CRDs (N-CRDs) and the other containing C-terminal CRDs (C-CRDs). The resulting binding surfaces at the opposite ends of the cylindrical trimer have the potential to cross-link cell surface or humoral carbohydrate ligands. The N-CRDs and C-CRDs of MsaFBP32 exhibit significant structural differences, suggesting that they recognize different glycans. Analysis of the carbohydrate binding sites provides the structural basis for the observed specificity of MsaFBP32 for simple carbohydrates and suggests that the N-CRD recognizes more complex fucosylated oligosaccharides and with a relatively higher avidity than the C-CRD. Modeling of MsaFBP32 complexed with fucosylated glycans that are widely distributed in prokaryotes and eukaryotes rationalizes the observation that binary tandem CRD F-type lectins function as opsonins by cross-linking "non-self" carbohydrate ligands and "self" carbohydrate ligands, such as sugar structures displayed by microbial pathogens and glycans on the surface of phagocytic cells from the host.

MeSH Categories: Amino Acid Sequence, Animals, Bass/genetics/*metabolism, Binding Sites, Crystallography, X-Ray, Fish Proteins/*chemistry/genetics/*metabolism, Fucose/metabolism, Lectins/*chemistry/genetics/*metabolism, Models, Molecular, Molecular Sequence Data, Protein Conformation, Protein Structure, Quaternary, Protein Structure, Tertiary, Sequence Homology, Amino Acid, Static Electricity

post to: CiteULike

Regulatory insertion removal restores maturation, stability and function of DeltaF508 CFTR.

Publication Date: 2010 Aug 13 PMID: 20561529
Authors: Aleksandrov, A. A. - Kota, P. - Aleksandrov, L. A. - He, L. - Jensen, T. - Cui, L. - Gentzsch, M. - Dokholyan, N. V. - Riordan, J. R.
Journal: J Mol Biol

The cystic fibrosis transmembrane conductance regulator (CFTR) epithelial anion channel is a large multidomain membrane protein that matures inefficiently during biosynthesis. Its assembly is further perturbed by the deletion of F508 from the first nucleotide-binding domain (NBD1) responsible for most cystic fibrosis. The mutant polypeptide is recognized by cellular quality control systems and is proteolyzed. CFTR NBD1 contains a 32-residue segment termed the regulatory insertion (RI) not present in other ATP-binding cassette transporters. We report here that RI deletion enabled F508 CFTR to mature and traffic to the cell surface where it mediated regulated anion efflux and exhibited robust single chloride channel activity. Long-term pulse-chase experiments showed that the mature DeltaRI/DeltaF508 had a T(1/2) of approximately 14 h in cells, similar to the wild type. RI deletion restored ATP occlusion by NBD1 of DeltaF508 CFTR and had a strong thermostabilizing influence on the channel with gating up to at least 40 degrees C. None of these effects of RI removal were achieved by deletion of only portions of RI. Discrete molecular dynamics simulations of NBD1 indicated that RI might indirectly influence the interaction of NBD1 with the rest of the protein by attenuating the coupling of the F508-containing loop with the F1-like ATP-binding core subdomain so that RI removal overcame the perturbations caused by F508 deletion. Restriction of RI to a particular conformational state may ameliorate the impact of the disease-causing mutation.

MeSH Categories: Adenosine Triphosphate/metabolism, Animals, Binding Sites/genetics, Biological Transport, Active, Cell Line, Cell Membrane/metabolism, Cricetinae, Cystic Fibrosis/genetics/metabolism, Cystic Fibrosis Transmembrane Conductance, Regulator/*chemistry/*genetics/metabolism, Humans, Mice, Models, Molecular, Molecular Dynamics Simulation, Mutant Proteins/chemistry/genetics/metabolism, Protein Interaction Domains and Motifs, Protein Processing, Post-Translational, Protein Stability, Recombinant Proteins/chemistry/genetics/metabolism, Sequence Deletion

post to: CiteULike

Recycling of the posttermination complexes of Mycobacterium smegmatis and Escherichia coli ribosomes using heterologous factors.

Publication Date: 2010 Sep 3 PMID: 20561528
Authors: Seshadri, A. - Singh, N. S. - Varshney, U.
Journal: J Mol Biol

In eubacteria, ribosome recycling factor (RRF) and elongation factor G (EFG) function together to dissociate posttermination ribosomal complexes. Earlier studies, using heterologous factors from Mycobacterium tuberculosis in Escherichia coli revealed that specific interactions between RRF and EFG are crucial for their function in ribosome recycling. Here, we used translation factors from E. coli, Mycobacterium smegmatis and M. tuberculosis, and polysomes from E. coli and M. smegmatis, and employed in vivo and in vitro experiments to further understand the role of EFG in ribosome recycling. We show that E. coli EFG (EcoEFG) recycles E. coli ribosomes with E. coli RRF (EcoRRF), but not with mycobacterial RRFs. Also, EcoEFG fails to recycle M. smegmatis ribosomes with either EcoRRF or mycobacterial RRFs. On the other hand, mycobacterial EFGs recycle both E. coli and M. smegmatis ribosomes with either of the RRFs. These observations suggest that EFG establishes distinct interactions with RRF and the ribosome to carry out ribosome recycling. Furthermore, the EFG chimeras generated by swapping domains between mycobacterial EFGs and EcoEFG suggest that while the residues needed to specify the EFG interaction with RRF are located in domains IV and V, those required to specify its interaction with the ribosome are located throughout the molecule.

post to: CiteULike

ATP binding, ATP hydrolysis, and protein dimerization are required for RecF to catalyze an early step in the processing and recovery of replication forks disrupted by DNA damage.

Publication Date: 2010 Aug 27 PMID: 20558179
Authors: Michel-Marks, E. - Courcelle, C. T. - Korolev, S. - Courcelle, J.
Journal: J Mol Biol

In Escherichia coli, the recovery of replication following disruption by UV-induced DNA damage requires the RecF protein and occurs through a process that involves stabilization of replication fork DNA, resection of nascent DNA to allow the offending lesion to be repaired, and reestablishment of a productive replisome on the DNA. RecF forms a homodimer and contains an ATP binding cassette ATPase domain that is conserved among eukaryotic SMC (structural maintenance of chromosome) proteins, including cohesin, condensin, and Rad50. Here, we investigated the functions of RecF dimerization, ATP binding, and ATP hydrolysis in the progressive steps involved in recovering DNA synthesis following disruption by DNA damage. RecF point mutations with altered biochemical properties were constructed in the chromosome. We observed that protein dimerization, ATP binding, and ATP hydrolysis were essential for maintaining and processing the arrested replication fork, as well as for restoring DNA synthesis. In contrast, stabilization of the RecF protein dimer partially protected the DNA at the arrested fork from degradation, although overall processing and recovery remained severely impaired.

MeSH Categories: Adenosine Triphosphatases/genetics/metabolism, Adenosine Triphosphate/*metabolism, Binding Sites, Catalysis, *DNA Damage, DNA Replication, DNA, Bacterial/metabolism, DNA-Binding Proteins/chemistry/genetics/*metabolism, Escherichia coli Proteins/chemistry/genetics/*metabolism, Hydrolysis, Protein Multimerization

post to: CiteULike

Intramembrane proteolysis of Mgm1 by the mitochondrial rhomboid protease is highly promiscuous regarding the sequence of the cleaved hydrophobic segment.

Publication Date: 2010 Aug 13 PMID: 20558178
Authors: Schafer, A. - Zick, M. - Kief, J. - Steger, M. - Heide, H. - Duvezin-Caubet, S. - Neupert, W. - Reichert, A. S.
Journal: J Mol Biol

Rhomboids are a family of intramembrane serine proteases that are conserved in bacteria, archaea, and eukaryotes. They are required for numerous fundamental cellular functions such as quorum sensing, cell signaling, and mitochondrial dynamics. Mitochondrial rhomboids form an evolutionarily distinct class of rhomboids. It is largely unclear how their activity is controlled and which substrate determinants are responsible for recognition and cleavage. We investigated these requirements for the mitochondrial rhomboid protease Pcp1 and its substrate Mgm1. In contrast to several other rhomboid proteases, Pcp1 does not require helix-breaking amino acids in the cleaved hydrophobic region of Mgm1, termed 'rhomboid cleavage region' (RCR). Even transmembrane segments of inner membrane proteins that are normally not processed by Pcp1 become cleavable when put in place of the authentic RCR of Mgm1. We further show that mutational alterations of a highly negatively charged region located C-terminally to the RCR led to a strong processing defect. Moreover, we show that the determinants required for Mgm1 processing by mitochondrial rhomboid protease are conserved during evolution, as PARL (the human ortholog of Pcp1) showed similar substrate requirements. These results suggest a surprising promiscuity of the mitochondrial rhomboid protease regarding the sequence requirements of the cleaved hydrophobic segment. We propose a working hypothesis on how the mitochondrial rhomboid protease can, despite this promiscuity, achieve a high specificity in recognizing Mgm1. This hypothesis relates to the exceptional biogenesis pathway of Mgm1.

MeSH Categories: Amino Acid Sequence, Binding Sites/genetics, Conserved Sequence, GTP-Binding Proteins/*chemistry/genetics/*metabolism, Humans, Hydrophobicity, Membrane Proteins/metabolism, Metalloproteases/metabolism, Mitochondrial Proteins/*chemistry/genetics/*metabolism, Models, Biological, Molecular Sequence Data, Mutagenesis, Site-Directed, Mutant Proteins/chemistry/genetics/metabolism, Protein Processing, Post-Translational, Recombinant Proteins/chemistry/genetics/metabolism, Saccharomyces cerevisiae/genetics/metabolism, Saccharomyces cerevisiae Proteins/*chemistry/genetics/*metabolism, Sequence Homology, Amino Acid, Serine Endopeptidases/*metabolism, Serine Proteases/*metabolism, Species Specificity, Substrate Specificity

post to: CiteULike

Crystal structure and functional analysis of the glutaminyl cyclase from Xanthomonas campestris.

Publication Date: 2010 Aug 20 PMID: 20558177
Authors: Huang, W. L. - Wang, Y. R. - Ko, T. P. - Chia, C. Y. - Huang, K. F. - Wang, A. H.
Journal: J Mol Biol

Glutaminyl cyclases (QCs) (EC 2.3.2.5) catalyze the formation of pyroglutamate (pGlu) at the N-terminus of many proteins and peptides, a critical step for the maturation of these bioactive molecules. Proteins having QC activity have been identified in animals and plants, but not in bacteria. Here, we report the first bacterial QC from the plant pathogen Xanthomonas campestris (Xc). The crystal structure of the enzyme was solved and refined to 1.44-A resolution. The structure shows a five-bladed beta-propeller and exhibits a scaffold similar to that of papaya QC (pQC), but with some sequence deletions and conformational changes. In contrast to the pQC structure, the active site of XcQC has a wider substrate-binding pocket, but its accessibility is modulated by a protruding loop acting as a flap. Enzyme activity analyses showed that the wild-type XcQC possesses only 3% QC activity compared to that of pQC. Superposition of those two structures revealed that an active-site glutamine residue in pQC is substituted by a glutamate (Glu(45)) in XcQC, although position 45 is a glutamine in most bacterial QC sequences. The E45Q mutation increased the QC activity by an order of magnitude, but the mutation E45A led to a drop in the enzyme activity, indicating the critical catalytic role of this residue. Further mutagenesis studies support the catalytic role of Glu(89) as proposed previously and confirm the importance of several conserved amino acids around the substrate-binding pocket. XcQC was shown to be weakly resistant to guanidine hydrochloride, extreme pH, and heat denaturations, in contrast to the extremely high stability of pQC, despite their similar scaffold. On the basis of structure comparison, the low stability of XcQC may be attributed to the absence of both a disulfide linkage and some hydrogen bonds in the closure of beta-propeller structure. These results significantly improve our understanding of the catalytic mechanism and extreme stability of type I QCs, which will be useful in further applications of QC enzymes.

post to: CiteULike

The glutamate effect on DNA binding by pol I DNA polymerases: osmotic stress and the effective reversal of salt linkage.

Publication Date: 2010 Aug 13 PMID: 20558176
Authors: Deredge, D. J. - Baker, J. T. - Datta, K. - Licata, V. J.
Journal: J Mol Biol

The significant enhancing effect of glutamate on DNA binding by Escherichia coli nucleic acid binding proteins has been extensively documented. Glutamate has also often been observed to reduce the apparent linked ion release (Deltan(ions)) upon DNA binding. In this study, it is shown that the Klenow and Klentaq large fragments of the Type I DNA polymerases from E. coli and Thermus aquaticus both display enhanced DNA binding affinity in the presence of glutamate versus chloride. Across the relatively narrow salt concentration ranges often used to obtain salt linkage data, Klenow displays an apparently decreased Deltan(ions) in the presence of Kglutamate, while Klentaq appears not to display an anion-specific effect on Deltan(ions). Osmotic stress experiments reveal that DNA binding by Klenow and Klentaq is associated with the release of approximately 500 to 600 waters in the presence of KCl. For both proteins, replacing chloride with glutamate results in a 70% reduction in the osmotic-stress-measured hydration change associated with DNA binding (to approximately 150-200 waters released), suggesting that glutamate plays a significant osmotic role. Measurements of the salt-DNA binding linkages were extended up to 2.5 M Kglutamate to further examine this osmotic effect of glutamate, and it is observed that a reversal of the salt linkage occurs above 800 mM for both Klenow and Klentaq. Salt-addition titrations confirm that an increase of [Kglutamate] beyond 1 M results in rebinding of salt-displaced polymerase to DNA. These data represent a rare documentation of a reversed ion linkage for a protein-DNA interaction (i.e., enhanced binding as salt concentration increases). Nonlinear linkage analysis indicates that this unusual behavior can be quantitatively accounted for by a shifting balance of ionic and osmotic effects as [Kglutamate] is increased. These results are predicted to be general for protein-DNA interactions in glutamate salts.

MeSH Categories: Base Sequence, Chlorides/metabolism, DNA Polymerase I/*metabolism, DNA, Bacterial/genetics/*metabolism, Escherichia coli/genetics/metabolism, Fluorescence Polarization, Glutamates/metabolism, Glutamic Acid/*metabolism, Kinetics, Osmotic Pressure, Potassium Chloride/metabolism, Salts/metabolism, Taq Polymerase/metabolism, Thermodynamics, Water/metabolism

post to: CiteULike

Folding and fibrillogenesis: clues from beta2-microglobulin.

Publication Date: 2010 Aug 13 PMID: 20558175
Authors: Rennella, E. - Corazza, A. - Giorgetti, S. - Fogolari, F. - Viglino, P. - Porcari, R. - Verga, L. - Stoppini, M. - Bellotti, V. - Esposito, G.
Journal: J Mol Biol

Renal failure impairs the clearance of beta(2)-microglobulin from the serum, with the result that this protein accumulates in joints under the form of amyloid fibrils. While the molecular mechanism leading to deposition of amyloid in vivo is not totally understood, some organic compounds, such as trifluoroethanol (TFE), are commonly used to promote the elongation of amyloid fibrils in vitro. This article gives some insights into the structural properties and the conformational states of beta(2)-microglobulin in the presence of TFE, using both the wild-type protein and the mutant Trp60Gly. The structure of the native state of the protein is rather insensitive to the presence of the alcohol, but the stability of this state is lowered in comparison to some other conformational states. In particular, a native-like folding intermediate is observed in the presence of moderate concentrations of TFE. Instead, at higher concentrations of the alcohol, the population of a disordered native-unlike state is dominant and correlates with the ability to elongate fibrils.

MeSH Categories: Amino Acid Substitution, Amyloid/chemistry, Humans, Microscopy, Electron, Transmission, Models, Molecular, Mutant Proteins/chemistry/genetics, Nuclear Magnetic Resonance, Biomolecular, Protein Conformation, Protein Denaturation, Protein Folding, Recombinant Proteins/chemistry/genetics, Thermodynamics, Trifluoroethanol, beta 2-Microglobulin/*chemistry/genetics/ultrastructure

post to: CiteULike

The structure of the FnIII Tandem A77-A78 points to a periodically conserved architecture in the myosin-binding region of titin.

Publication Date: 2010 Sep 3 PMID: 20542041
Authors: Bucher, R. M. - Svergun, D. I. - Muhle-Goll, C. - Mayans, O.
Journal: J Mol Biol

Titin is a large intrasarcomeric protein that, among its many roles in muscle, is thought to modulate the in vivo assembly of the myosin motor filament. This is achieved through the molecular template properties of its A-band region, which is composed of fibronectin type III (FnIII) and immunoglobulin (Ig) domains organized into characteristic 7-domain (D-zone) and 11-domain (C-zone) superrepeats. Currently, there is little knowledge on the structural details of this region of titin. Here we report the conformational characterization of three FnIII tandems, A77-A78, A80-A82, and A84-A86, which are components of the representative fourth C-zone superrepeat. The structure of A77-A78 has been elucidated by X-ray crystallography to 1.65 A resolution, while low-resolution models of A80-A82 and A84-A86 have been calculated using small-angle X-ray scattering. A77-A78 adopts an extended "up-down" domain arrangement, where domains are connected by a hydrophilic three-residue linker sequence. The linker is embedded in a rich network of polar contacts at the domain interface that results in a stiff molecular conformation. The models of A80-A82 and A84-A86, which contain hydrophobic six-residue-long interdomain linkers, equally showed elongated molecular shapes, but with slightly coiled or zigzagged conformations. Small-angle X-ray scattering data further suggested that the long linkers do not result in a noticeable increase in molecular flexibility but lead to semibent domain arrangements. Our findings indicate that the structural characteristics of FnIII tandems from A-band titin contrast markedly with those of poly-Ig tandems from the elastic I-band, which exhibit domain interfaces depleted of interactions and compliant conformations. Furthermore, the analysis of sequence conservation in FnIII domains from A-band titin points to the existence of conformationally defined interfaces at specific superrepeat positions, possibly leading to a periodic and locally ordered architecture supporting the molecular scaffold properties of this region of titin.

post to: CiteULike

Evolved Lactococcus lactis strains for enhanced expression of recombinant membrane proteins.

Publication Date: 2010 Aug 6 PMID: 20542040
Authors: Linares, D. M. - Geertsma, E. R. - Poolman, B.
Journal: J Mol Biol

The production of complex multidomain (membrane) proteins is a major hurdle in structural genomics and a generic approach for optimizing membrane protein expression is still lacking. We have devised a selection method to isolate mutant strains with improved functional expression of recombinant membrane proteins. By fusing green fluorescent protein and an erythromycin resistance marker (ErmC) to the C-terminus of a target protein, one simultaneously selects for variants with enhanced expression (increased erythromycin resistance) and correct folding (green fluorescent protein fluorescence). Three evolved hosts, displaying 2- to 8-fold increased expression of a plethora of proteins, were fully sequenced and shown to carry single-site mutations in the nisK gene. NisK is the sensor protein of a two-component regulatory system that directs nisin-A-mediated expression. The levels of recombinant membrane proteins were increased in the evolved strains, and in some cases their folding states were improved. The generality and simplicity of our approach allow rapid improvements of protein production yields by directed evolution in a high-throughput way.

MeSH Categories: Cloning, Molecular, Directed Molecular Evolution, Gene Expression Regulation, Bacterial/*genetics, Genes, Bacterial/genetics/physiology, Genes, Reporter, Genome, Bacterial, Green Fluorescent Proteins/genetics/metabolism, High-Throughput Screening Assays, Lactococcus lactis/*genetics/*metabolism, Membrane Proteins/*genetics/metabolism, Organisms, Genetically Modified, Protein Folding, Recombinant Fusion Proteins/chemistry/genetics/metabolism, Recombinant Proteins/*genetics/metabolism, Species Specificity, Up-Regulation/genetics

post to: CiteULike

The unfolded state of the murine prion protein and properties of single-point mutants related to human prion diseases.

Publication Date: 2010 Aug 6 PMID: 20541558
Authors: Gerum, C. - Schlepckow, K. - Schwalbe, H.
Journal: J Mol Biol

The prion protein can exist both in a normal cellular isoform and in a pathogenic conformational isoform. The latter is responsible for the development of different neurodegenerative diseases, for example Creutzfeldt-Jakob disease or fatal familial insomnia. To convert the native benign state of the protein into a highly ordered fibrillar aggregate, large-scale rearrangements of the tertiary structure are necessary during the conversion process and intermediates that are at least partially unfolded are present during fibril formation. In addition to the sporadic conversion into the pathogenic isoform, more than 20 familial diseases are known that are caused by single point mutations increasing the probability of aggregation and neurodegeneration. Here, we demonstrate that the chemically denatured states of the mouse and human prion proteins have very similar structural and dynamic characteristics. Initial studies on the single point mutants E196K, F198S, V203I and R208H of the oxidized mouse construct, which are related to human prion diseases, reveal significant differences in the rate of aggregation. Aggregation for mutants V203I and R208H is slower than it is for the wild type, and the constructs E196K and F198S show accelerated aggregation. These differences in aggregation behaviour are not correlated with the thermal stability of the mutants, indicating different mechanisms promoting the conformational conversion process.

MeSH Categories: Amino Acid Substitution/genetics/physiology, Animals, Chemical Precipitation, Humans, Mice, Mutagenesis, Site-Directed, Mutant Proteins/chemistry/genetics/metabolism/*physiology, *Point Mutation/physiology, Prion Diseases/*genetics/metabolism, Prions/*chemistry/*genetics/metabolism, Protein Conformation, Protein Folding, Protein Stability, Species Specificity, Thermodynamics

post to: CiteULike

SDS-induced fibrillation of alpha-synuclein: an alternative fibrillation pathway.

Publication Date: 2010 Aug 6 PMID: 20540950
Authors: Giehm, L. - Oliveira, C. L. - Christiansen, G. - Pedersen, J. S. - Otzen, D. E.
Journal: J Mol Biol

A structural investigation of the sodium dodecyl sulfate (SDS)-induced fibrillation of alpha-synuclein (alphaSN), a 140-amino-acid protein implicated in Parkinson's disease, has been performed. Spectroscopic analysis has been combined with isothermal titration calorimetry, small-angle X-ray scattering, and transmission electron microscopy to elucidate a fibrillation pathway that is remarkably different from the fibrillation pathway in the absence of SDS. Fibrillation occurs most extensively and most rapidly (starting within 45 min) under conditions where 12 SDS molecules are bound per alphaSN molecule, which is also the range where SDS binding is associated with the highest enthalpy. Fibrillation is only reduced in proportion to the fraction of SDS below 25 mol% SDS in mixed surfactant mixtures with nonionic surfactants and is inhibited by formation of bulk micelles and induction of alpha-helical structure. In this fibrillogenic complex, 4 alphaSN molecules initially associate with 40-50 SDS molecules to form a shared micelle that gradually grows in size. The complex initially exhibits a mixture of random coil and alpha-helix, but incubation results in a structural conversion into beta-sheet structure and concomitant formation of thioflavin-T-binding fibrils over a period of several hours. Based on small-angle X-ray scattering, the aggregates elongate as a beads-on-a-string structure in which individual units of ellipsoidal SDS-alphaSN are bridged by strings of the protein, so that aggregates nucleate around the surface of protein-stabilized micelles. Thus, fibrillation in this case occurs by a process of continuous accretion rather than by the rate-limiting accumulation of a distinct nucleus. The morphology of the SDS-induced fibrils does not exhibit the classical rod-like structures formed by alphaSN when aggregated by agitation in the absence of SDS. The SDS-induced fibrils have a flexible worm-like appearance, which can be converted into classical straight fibrils by continuous agitation. SDS-induced fibrillation represents an alternative and highly reproducible mechanism for fibrillation where protein association is driven by the formation of shared micelles, which subsequently allows the formation of beta-sheet structures that presumably link individual micelles. This illustrates that protein fibrillation may occur by remarkably different mechanisms, testifying to the versatility of this process.

MeSH Categories: Amyloid/chemical synthesis/chemistry/drug effects/*metabolism, Circular Dichroism, Humans, Metabolic Networks and Pathways/drug effects, Micelles, Models, Biological, Protein Binding, Protein Conformation, Protein Multimerization/*drug effects, Recombinant Proteins/chemistry/metabolism, Scattering, Small Angle, Sodium Dodecyl Sulfate/*pharmacology, Surface-Active Agents/pharmacology, X-Rays, alpha-Synuclein/*chemistry/drug effects/*metabolism

post to: CiteULike

Stability and CDR composition biases enrich binder functionality landscapes.

Publication Date: 2010 Aug 6 PMID: 20540948
Authors: Hackel, B. J. - Ackerman, M. E. - Howland, S. W. - Wittrup, K. D.
Journal: J Mol Biol

The rugged protein sequence-function landscape complicates efforts, both in nature and in the laboratory, to evolve protein function. Protein library diversification must strike a balance between sufficient variegation to thoroughly sample alternative functionality versus the probability of mutant destabilization below an expressible threshold. In this work, we explore the sequence-function landscape in the context of screening for molecular recognition from an Ig scaffold library. The fibronectin type III domain is used to explore the impact of two sequence diversification strategies: (a) partial wild-type conservation at structurally important positions within the paratope region and (b) tailored amino acid composition mimicking antibody binding-site composition at putative paratope positions. Structurally important positions within the paratope region were identified through stability, structural, and phylogenetic analyses and partially or fully conserved in sequence. To achieve tailored antibody-like diversity, we designed a set of skewed nucleotide mixtures yielding codons approximately matching the distribution observed in antibody complementarity-determining regions without incurring the expense of triphosphoramidite-based construction. These design elements were explored via comparison of three library designs: a random library, a library with wild-type bias in the DE loop only and tyrosine-serine diversity elsewhere, and a library with wild-type bias at 11 positions and the antibody-inspired amino acid distribution. Using pooled libraries for direct competition in a single tube, selection and maturation of binders to seven targets yielded 19 of 21 clones that originated from the structurally biased, tailored-diversity library design. Sequence analysis of the selected clones supports the importance of both tailored compositional diversity and structural bias. In addition, selection of both well and poorly expressed clones from two libraries further elucidated the impact of structural bias.

MeSH Categories: Amino Acid Sequence, Animals, Antibodies/*chemistry/*metabolism, Antibody Affinity, Binding, Competitive, Cells, Cultured, Complementarity Determining Regions/*chemistry/*metabolism, Humans, Models, Molecular, Peptide Library, Phylogeny, Protein Binding/physiology, Protein Engineering/methods, Protein Stability, Saccharomyces cerevisiae, Sequence Analysis, Protein/methods, Structure-Activity Relationship

post to: CiteULike

Solution structure of histone chaperone ANP32B: interaction with core histones H3-H4 through its acidic concave domain.

Publication Date: 2010 Aug 6 PMID: 20538007
Authors: Tochio, N. - Umehara, T. - Munemasa, Y. - Suzuki, T. - Sato, S. - Tsuda, K. - Koshiba, S. - Kigawa, T. - Nagai, R. - Yokoyama, S.
Journal: J Mol Biol

Eukaryotic gene expression is regulated by histone deposition onto and eviction from nucleosomes, which are mediated by several chromatin-modulating factors. Among them, histone chaperones are key factors that facilitate nucleosome assembly. Acidic nuclear phosphoprotein 32B (ANP32B) belongs to the ANP32 family, which shares N-terminal leucine-rich repeats (LRRs) and a C-terminal variable anionic region. The C-terminal region functions as an inhibitor of histone acetylation, but the functional roles of the LRR domain in chromatin regulation have remained elusive. Here, we report that the LRR domain of ANP32B possesses histone chaperone activity and forms a curved structure with a parallel beta-sheet on the concave side and mostly helical elements on the convex side. Our analyses revealed that the interaction of ANP32B with the core histones H3-H4 occurs on its concave side, and both the acidic and hydrophobic residues that compose the concave surface are critical for histone binding. These results provide a structural framework for understanding the functional mechanisms of acidic histone chaperones.

MeSH Categories: Amino Acid Sequence, Amino Acids, Acidic/chemistry/*metabolism, Hela Cells, Histone Chaperones/chemistry/metabolism, Histones/*metabolism, Humans, Hydrophobicity, Models, Molecular, Molecular Sequence Data, Nuclear Proteins/*chemistry/*metabolism, Protein Binding, Protein Interaction Mapping, Protein Structure, Tertiary/drug effects, Sequence Homology, Amino Acid, Solutions/pharmacology

post to: CiteULike

K65R and K65A substitutions in HIV-1 reverse transcriptase enhance polymerase fidelity by decreasing both dNTP misinsertion and mispaired primer extension efficiencies.

Publication Date: 2010 Aug 6 PMID: 20538005
Authors: Garforth, S. J. - Domaoal, R. A. - Lwatula, C. - Landau, M. J. - Meyer, A. J. - Anderson, K. S. - Prasad, V. R.
Journal: J Mol Biol

Lys65 residue, in the fingers domain of human immunodeficiency virus reverse transcriptase (RT), interacts with incoming dNTP in a sequence-independent fashion. We showed previously that a 5-amino-acid deletion spanning Lys65 and a K65A substitution both enhanced the fidelity of dNTP insertion. We hypothesized that the Lys65 residue enhances dNTP misinsertion via interactions with the gamma-phosphate of the incoming dNTP. We now examine this hypothesis in pre-steady-state kinetic studies using wild-type human immunodeficiency virus-1 RT and two substitution mutants, K65A and K65R. K65R mutation did not greatly increase misinsertion fidelity, but K65A mutation led to higher incorporation fidelity. For a misinsertion to become a permanent error, it needs to be accompanied by the extension of the mispaired terminus thus formed. Both mutants and the wild-type enzyme discriminated against the mismatched primer at the catalytic step (k(pol)). Additionally, K65A and K65R mutants displayed a further decrease in mismatch extension efficiency, primarily at the level of dNTP binding. We employed hydroxyl radical footprinting to determine the position of the RT on the primer/template. The wild-type and Lys65-substituted enzymes occupied the same position at the primer terminus; the presence of a mismatched primer terminus caused all three enzymes to be displaced to a -2 position relative to the primer 3' end. In the context of an efficiently extended mismatched terminus, the presence of the next complementary nucleotide overcame the displacement, resulting in a complex resembling the matched terminus. The results are consistent with the observed reduction in k(pol) in mispaired primer extension being due to the position of the enzyme at a mismatched terminus. Our work shows the influence of the stabilizing interactions of Lys65 with the incoming dNTP on two different aspects of polymerase fidelity.

MeSH Categories: Alanine/genetics, *Amino Acid Substitution/genetics/physiology, Arginine/genetics, DNA Mismatch Repair/*genetics, DNA Primers/genetics, DNA-Directed DNA Polymerase/genetics/metabolism, HIV Reverse Transcriptase/*genetics/*metabolism/physiology, Lysine/genetics, Models, Biological, Mutagenesis, Insertional/*genetics, Nucleotides/metabolism, Structure-Activity Relationship, Templates, Genetic, Up-Regulation

post to: CiteULike

Architecture of the Tn7 posttransposition complex: an elaborate nucleoprotein structure.

Publication Date: 2010 Aug 13 PMID: 20538004
Authors: Holder, J. W. - Craig, N. L.
Journal: J Mol Biol

Four transposition proteins encoded by the bacterial transposon Tn7, TnsA, TnsB, TnsC, and TnsD, mediate its site- and orientation-specific insertion into the chromosomal site attTn7. To establish which Tns proteins are actually present in the transpososome that executes DNA breakage and joining, we have determined the proteins present in the nucleoprotein product of transposition, the posttransposition complex (PTC), using fluorescently labeled Tns proteins. All four required Tns proteins are present in the PTC in which we also find that the Tn7 ends are paired by protein-protein contacts between Tns proteins bound to the ends. Quantification of the relative amounts of the fluorescent Tns proteins in the PTC indicates that oligomers of TnsA, TnsB, and TnsC mediate Tn7 transposition. High-resolution DNA footprinting of the DNA product of transposition attTn7Colon, two colonsTn7 revealed that about 350 bp of DNA on the transposon ends and on attTn7 contact the Tns proteins. All seven binding sites for TnsB, the component of the transposase that specifically binds the ends and mediates 3' end breakage and joining, are occupied in the PTC. However, the protection pattern of the sites closest to the Tn7 ends in the PTC are different from that observed with TnsB alone, likely reflecting the pairing of the ends and their interaction with the target nucleoprotein complex necessary for activation of the breakage and joining steps. We also observe extensive protection of the attTn7 sequences in the PTC and that alternative DNA structures in substrate attTn7 that are imposed by TnsD are maintained in the PTC.

MeSH Categories: Base Sequence, Binding Sites, DNA Footprinting, DNA Transposable Elements/*genetics, DNA, Bacterial/chemistry/*genetics/metabolism, Escherichia coli/genetics/metabolism, Escherichia coli Proteins/*chemistry/*genetics/metabolism, Microscopy, Atomic Force, Molecular Sequence Data, Molecular Structure, Multiprotein Complexes/chemistry, Nucleoproteins/*chemistry/*genetics/metabolism, Transposases/chemistry/genetics/metabolism

post to: CiteULike