Protein Science

In this issue.

Publication Date: 2012 Jun PMID: 22605670
Authors:
Journal: Protein Sci

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Designing specific protein-protein interactions using computation, experimental library screening, or integrated methods.

Publication Date: 2012 May 16 PMID: 22593041
Authors: Chen, T. S. - Keating, A. E.
Journal: Protein Sci

Given the importance of protein-protein interactions for nearly all biological processes, the design of protein affinity reagents for use in research, diagnosis or therapy is an important endeavor. Engineered proteins would ideally have high specificities for their intended targets, but achieving interaction specificity by design can be challenging. There are two major approaches to protein design or re-design. Most commonly, proteins and peptides are engineered using experimental library screening and/or in vitro evolution. An alternative approach involves using protein structure and computational modeling to rationally choose sequences predicted to have desirable properties. Computational design has successfully produced novel proteins with enhanced stability, desired interactions and enzymatic function. Here we review the strengths and limitations of experimental library screening and computational structure-based design, giving examples where these methods have been applied to designing protein interaction specificity. We highlight recent studies that demonstrate strategies for combining computational modeling with library screening. The computational methods provide focused libraries predicted to be enriched in sequences with the properties of interest. Such integrated approaches represent a promising way to increase the efficiency of protein design and to engineer complex functionality such as interaction specificity.

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Age-dependent deamidation of glutamine residues in human gammaS crystallin: Deamidation and unstructured regions.

Publication Date: 2012 May 16 PMID: 22593035
Authors: Hooi, M. Y. - Raftery, M. J. - Truscott, R. J.
Journal: Protein Sci

Human aging is associated with the deterioration of long-lived proteins. Gradual cumulative modifications to the life-long proteins of the lens may ultimately be responsible for the pronounced alterations to the optical and physical properties that characterize lenses from older people. gammaS crystallin, a major human lens protein, is known to undergo several age-dependent changes. Using proteomic techniques, a site of deamidation involving glutamine 92 has been characterized and its time course established. The proportion of deamidation increased from birth to teen-age years and then plateaud. Deamidation at this site increased again in the eighth decade of life. There was no significant difference in the extent of deamidation between cataract and age-matched normal lenses. Gln 92 is located in the linker region between the two domains and the introduction of a negative charge at this site may alter the interaction between the two regions of the protein. Gln 170, that is located in another unstructured part of gammaS crystallin, showed a similar deamidation profile to that of Gln 92. Since the other Gln residues in betasheet regions of gammaS crystallin appear to remain as amides, modification of Gln 92 and Gln 170 thus conforms to a pattern whereby deamidation is localized to the unstructured regions of long-lived proteins.

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Sequence-dependent backbone dynamics of a viral fusogen transmembrane helix.

Publication Date: 2012 May 16 PMID: 22593029
Authors: Stelzer, W. - Langosch, D.
Journal: Protein Sci

The transmembrane domains of membrane fusogenic proteins are known to contribute to lipid bilayer mixing as indicated by mutational studies and functional reconstitution of peptide mimics. Here, we demonstrate that mutations of a GxxxG motif or of Ile residues, that were previously shown to compromise the fusogenicity of the Vesicular Stomatitis virus G-protein transmembrane helix, reduce its backbone dynamics as determined by deuterium/hydrogen-exchange kinetics. Thus, the backbone dynamics of these helices may be linked to their fusogenicity which is consistent with the known over-representation of Gly and Ile in viral fusogen transmembrane helices.

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Sensing the messenger: The diverse ways that bacteria signal through c-di-GMP.

Publication Date: 2012 May 16 PMID: 22593024
Authors: Krasteva, P. V. - Giglio, K. M. - Sondermann, H.
Journal: Protein Sci

An intracellular second messenger unique to bacteria, c-di-GMP has gained appreciation as a key player in adaptation and virulence strategies, such as biofilm formation, persistence, and cytotoxicity. Diguanylate cyclases containing GGDEF domains and phosphodiesterases containing either EAL or HD-GYP domains have been identified as the enzymes controlling intracellular c-di-GMP levels, yet little is known regarding signal transmission and the sensory targets for this signaling molecule. Although limited in number, identified c-di-GMP receptors in bacteria are characterized by prominent diversity and multilevel impact. In addition, c-di-GMP has been shown to have immunomodulatory effects in mammals and several eukaryotic c-di-GMP sensors have been proposed. The structural biology of c-di-GMP receptors is a rapidly developing field of research, which holds promise for the development of novel therapeutics against bacterial infections. In this review, we highlight recent advances in identifying bacterial and eukaryotic c-di-GMP signaling mechanisms and emphasize the need for mechanistic structure-function studies on confirmed signaling targets.

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The dynamical response of hen egg white lysozyme to the binding of a carbohydrate ligand.

Publication Date: 2012 May 16 PMID: 22593013
Authors: Moorman, V. R. - Valentine, K. G. - Wand, A. J.
Journal: Protein Sci

It has become clear that the binding of small and large ligands to proteins can invoke significant changes in side chain and main chain motion in the fast ps-ns timescale. Recently, the use of a "dynamical proxy" has indicated that changes in these motions often reflect significant changes in conformational entropy. These entropic contributions are sometimes of the same order as the total entropy of binding. Thus it is important to understand the connections amongst motion between the manifold of states accessible to the native state of proteins, the corresponding entropy and how this impacts the overall energetics of protein function. The interaction of proteins with carbohydrate ligands is central to a range of biological functions. Here we examine a classic carbohydrate interaction with an enzyme: the binding of wild-type hen egg white lysozyme (HEWL) to the natural, competitive inhibitor chitotriose. Using NMR relaxation experiments, backbone amide and side chain methyl axial order parameters were obtained across apo and chitotriose-bound HEWL. Upon binding, changes in the apparent amplitude of ps-ns main chain and side chain motions are seen across the protein. Indeed, binding of chitotriose renders a large contiguous fraction of HEWL effectively completely rigid. Changes in methyl flexibility are most pronounced closest to the binding site, but average to only a small overall change in the dynamics across the protein. The corresponding change in conformational entropy is unfavorable and estimated to be a significant fraction of the total binding entropy.

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Mechanism of zinc-mediated inhibition of caspase-9.

Publication Date: 2012 May 9 PMID: 22573662
Authors: Huber, K. - Hardy, J. A.
Journal: Protein Sci

Zinc-mediated inhibition is implicated in global caspase regulation, with relief of zinc-mediated inhibition central to both small-molecule and natively-induced caspase activation. As an initiator, caspase-9 regulates the upstream stages of the apoptotic caspase cascade, making it a critical control point. Here we identify two distinct zinc-binding sites on caspase-9. The first site, composed of H237, C239, C287 includes the active site dyad and is primarily responsible for zinc-mediated inhibition. The second binding site at C272 plays is distal from the active site. Given the amino-acid conservation in both regions, these sites appear to be present across the caspase family underscoring the importance of zinc-mediated regulation of this class of enzymes.

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How cells process information: Quantification of spatiotemporal signaling dynamics.

Publication Date: 2012 May 9 PMID: 22573643
Authors: Ganesan, A. - Zhang, J.
Journal: Protein Sci

Arguably, one of the foremost distinctions between life and non-living matter is the ability to sense environmental changes and respond appropriately - an ability that is invested in every living cell. Within a single cell, this function is largely carried out by networks of signaling molecules. However, the details of how signaling networks help cells make complicated decisions are still not clear. For instance, how do cells read graded, analog stress signals but convert them into digital live-or-die responses? The answer to such questions may originate from the fact that signaling molecules are not static but dynamic entities, changing in numbers over time and space. In the past two decades, researchers have been able to experimentally monitor signaling dynamics and use mathematical techniques to quantify and abstract general principles of how cells process information. In this review, we first introduce and discuss various experimental and computational methodologies that have been used to study signaling dynamics. We then discuss the different types of temporal dynamics such as oscillations and bistability that can be exhibited by signaling systems and highlight studies that have investigated such dynamics in physiological settings. Finally, we illustrate the role of spatial compartmentalization in regulating cellular responses with examples of second messenger signaling in cardiac myocytes.

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N-terminal acetylation of alpha-synuclein induces increased transient helical propensity and decreased aggregation rates in the intrinsically disordered monomer.

Publication Date: 2012 May 9 PMID: 22573613
Authors: Kang, L. - Moriarty, G. M. - Woods, L. A. - Ashcroft, A. E. - Radford, S. E. - Baum, J.
Journal: Protein Sci

The conformational properties of soluble alpha-synuclein, the primary protein found in patients with Parkinson's disease, are thought to play a key role in the structural transition to amyloid fibrils. In this work, we report that recombinant 100% N-terminal acetylated alpha-synuclein purified under mild physiological conditions presents as a primarily monomeric protein and that the N-terminal acetyl group affects the transient secondary structure and fibril assembly rates of the protein. Residue-specific NMR chemical shift analysis indicates substantial increase in transient helical propensity in the first twelve N-terminal residues, as well as smaller long-range changes in residues 28 to 31, 43 to 46 and 50 to 66: regions in which the three familial mutations currently known to be causative of early onset disease are found. In addition we show that the N-terminal acetylated protein forms fibrils that are morphologically similar to those formed from non-acetylated alpha-synuclein, but that their growth rates are slower. Our results highlight that N-terminal acetylation does not form significant numbers of dimers, tetramers or higher molecular weight species but does alter the conformational distributions of monomeric alpha-synuclein species in regions known to be important in metal binding, in association with membranes, and in regions known to affect fibril formation rates.

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Transient interactions of a slow-folding protein with the Hsp70 chaperone machinery.

Publication Date: 2012 May 1 PMID: 22549943
Authors: Sekhar, A. - Santiago, M. - Lam, H. N. - Lee, J. H. - Cavagnero, S.
Journal: Protein Sci

Most known proteins have at least one local Hsp70 chaperone binding site. Does this mean that all proteins interact with Hsp70 as they fold? This study makes an initial step to address the above question by examining the interaction of the E.coli Hsp70 chaperone (known as DnaK) and its co-chaperones DnaJ and GrpE with a slow-folding E.coli substrate, RNase H(D) . Importantly, this protein is a non-obligatory client, and it is able to fold in vitro even in the absence of chaperones. We employ stopped-flow mixing, chromatography and activity assays to analyze the kinetic perturbations induced by DnaK/DnaJ/GrpE (K/J/E) on the folding of RNase H(D) . We find that K/J/E slows down RNase H(D) 's apparent folding, consistent with the presence of transient chaperone-substrate interactions. On the other hand, kinetic retardation is moderate for this slow-folding client and it is expected to be even smaller for faster-folding substrates. Given that the interaction of folding-competent substrates such as RNase H(D) with the K/J/E chaperone system is relatively short-lived, it does not significantly interfere with the timely production of folded biologically active substrate. The above mode of action is important because it preserves K/J/E bioavailability, enabling this chaperone system to act primarily on assisting the folding of other misfolded and (or) aggregation-prone cellular proteins that are unable to fold independently. When refolding is carried out in the presence of K/J and absence of the nucleotide exchange factor GrpE, some of the substrate population becomes trapped as a chaperone-bound partially unfolded state.

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Structural, kinetic, and thermodynamic studies of specificity designed HIV-1 protease.

Publication Date: 2012 May 1 PMID: 22549928
Authors: Alvizo, O. - Mittal, S. - Mayo, S. L. - Schiffer, C. A.
Journal: Protein Sci

HIV-1 protease recognizes and cleaves more than 12 different substrates leading to viral maturation. While these substrates share no conserved motif, they are specifically selected for and cleaved by protease during viral life cycle. Drug resistant mutations evolve within the protease that compromise inhibitor binding but allow the continued recognition of all these substrates. While the substrate envelope defines a general shape for substrate recognition, successfully predicting the determinants of substrate binding specificity would provide additional insights into the mechanism of altered molecular recognition in resistant proteases. We designed a variant of HIV protease with altered specificity using positive computational design methods and validated the design using x-ray crystallography and enzyme biochemistry. The engineered variant, Pr3 (A28S/D30F/G48R), was designed to preferentially bind to one out of three of HIV protease's natural substrates; RT-RH over p2-NC and CA-p2. In kinetic assays, RT-RH binding specificity for Pr3 increased three-fold compared to the wild-type (WT), which was further confirmed by isothermal titration calorimetry. Crystal structures of WT protease and the designed variant in complex with RT-RH, CA-p2 and p2-NC were determined. Structural analysis of the designed complexes revealed that one of the engineered substitutions (G48R) potentially stabilized heterogeneous flap conformations, thereby facilitating alternate modes of substrate binding. Our results demonstrate that while substrate specificity could be engineered in HIV protease, the structural pliability of protease restricted the propagation of interactions as predicted. These results offer new insights into the plasticity and structural determinants of substrate binding specificity of the HIV-1 protease.

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The bacterial outer membrane beta-barrel assembly machinery.

Publication Date: 2012 Jun PMID: 22549918
Authors: Kim, K. H. - Aulakh, S. - Paetzel, M.
Journal: Protein Sci

beta-Barrel proteins found in the outer membrane of Gram-negative bacteria serve a variety of cellular functions. Proper folding and assembly of these proteins are essential for the viability of bacteria and can also play an important role in virulence. The beta-barrel assembly machinery (BAM) complex, which is responsible for the proper assembly of beta-barrels into the outer membrane of Gram-negative bacteria, has been the focus of many recent studies. This review summarizes the significant progress that has been made toward understanding the structure and function of the bacterial BAM complex.

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Template-based structure prediction and classification of transcription factors in Arabidopsis thaliana.

Publication Date: 2012 Jun PMID: 22549903
Authors: Lu, T. - Yang, Y. - Yao, B. - Liu, S. - Zhou, Y. - Zhang, C.
Journal: Protein Sci

Transcription factors (TFs) play important roles in plants. However, there is no systematic study of their structures and functions of most TFs in plants. Here, we performed template-based structure prediction for all TFs in Arabidopsis thaliana, with their full-length sequences as well as C-terminal and N-terminal regions. A total of 2918 model structures were obtained with a high confidence score. We find that TF families employ only a smaller number of templates for DNA-binding domains (DBD) but a diverse number of templates for transcription regulatory domains (TRD). Although TF families are classified according to DBD, their sizes have a significant correlation with the number of unique non-DNA-binding templates employed in the family (Pearson correlation coefficient of 0.74). That is, the size of TF family is related to its functional diversity. Network analysis reveals new connections between TF families based on shared TRD or DBD templates; 81% TF families share DBD and 67% share TRD templates. Two large fully connected family clusters in this network are observed along with 69 island families. In addition, 25 genes with unknown functions are found to be DNA-binding and/or TF factors according to predicted structures. This work provides a global view of the classification of TFs based on their DBD or TRD templates, and hence, a deeper understanding of DNA-binding and regulatory functions from structural perspective. All structural models of TFs are deposited in the online database for public usage at http://sysbio.unl.edu/AthTF.

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Structure based optimization of designed armadillo-repeat proteins.

Publication Date: 2012 Apr 27 PMID: 22544642
Authors: Madhurantakam, C. - Varadamsetty, G. - Grutter, M. G. - Pluckthun, A. - Mittl, P. R.
Journal: Protein Sci

The armadillo domain is a right-handed super-helix of repeating units composed of three alpha-helices each. Armadillo repeat proteins (ArmRPs) are frequently involved in protein-protein interactions, because of their modular recognition of extended peptide regions they can serve as templates for the design of artificial peptide binding scaffolds. Based on sequential and structural analyses, different consensus designed ArmRPs were synthesized and show high thermodynamic stabilities, compared to naturally occurring ArmRPs. We determined the crystal structures of four full-consensus ArmRPs with 3 or 4 identical internal repeats and two different designs for the N- and C-caps. The crystal structures were refined at resolutions ranging from 1.80 to 2.50 A for the above mentioned designs. A redesign of our initial caps was required to obtain well diffracting crystals. However, the structures with the redesigned caps caused domain swapping events between the N-caps. To prevent this domain swap, 9 and 6 point mutations were introduced in the N- and C-caps, respectively. Structural and biophysical analysis showed that this subsequent redesign of the N-cap prevented domain swapping and improved the thermodynamic stability of the proteins. We systematically investigated the best cap combinations. We conclude that designed ArmRPs with optimized caps are intrinsically stable and well-expressed monomeric proteins and that the high-resolution structures provide excellent structural templates for the continuation of the design of sequence-specific modular peptide recognition units based on armadillo repeats.

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Mycobacterium thermoresistibile as a source of thermostable orthologs of mycobacterium tuberculosis proteins.

Publication Date: 2012 Apr 27 PMID: 22544630
Authors: Edwards, T. E. - Liao, R. - Phan, I. - Myler, P. J. - Grundner, C.
Journal: Protein Sci

The genus Mycobacterium comprises major human pathogens such as the causative agent of tuberculosis, Mycobacterium tuberculosis (Mtb), and many environmental species. Tuberculosis claims approximately 1.5 million lives every year, and drug resistant strains of Mtb are rapidly emerging. To aid the development of new tuberculosis drugs, major efforts are currently under way to determine crystal structures of Mtb drug targets and proteins involved in pathogenicity. However, a major obstacle to obtaining crystal structures is the generation of well-diffracting crystals. Proteins from thermophiles can have better crystallization and diffraction properties than proteins from mesophiles, but their sequences and structures are often divergent. Here, we establish a thermophilic mycobacterial model organism, Mycobacterium thermoresistibile (Mth), for the study of Mtb proteins. Mth tolerates higher temperatures than Mtb or other environmental mycobacteria such as M. smegmatis. Mth proteins are on average more soluble than Mtb proteins, and comparison of the crystal structures of two pairs of orthologous proteins reveals nearly identical folds, indicating that Mth structures provide good surrogates for Mtb structures. This study introduces a thermophile as a source of protein for the study of a closely related human pathogen and marks a new approach to solving challenging mycobacterial protein structures.

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X-interface is not the explanation for the slow disassembly of N-cadherin dimers in the apo state.

Publication Date: 2012 Apr 27 PMID: 22544613
Authors: Vunnam, N. - Pedigo, S.
Journal: Protein Sci

In spite of structural similarities Epithelial- (E-) and Neural- (N-) cadherins are expressed at two types of synapses and differ significantly in dimer disassembly kinetics. Recent studies suggested that the formation of an X-dimer intermediate in E-cadherin is the key requirement for rapid disassembly of the adhesive dimer (Harrison, et al (2010) and Hong, et al (2011)). The X-interface in E-cadherin involves three non-covalent interactions, none of which is conserved in N-cadherin. Dimer disassembly is slow at low calcium concentration in N-cadherin, which may be due to the differences in the X-interface residues. To investigate the origin of the slow disassembly kinetics we introduced three point mutations into N-cadherin to provide the opportunity for the formation of X-interface interactions. Spectroscopic studies showed that the triple mutation did not affect the stability or the calcium-binding affinity of the X-enabled N-cadherin mutant. Analytical Size Exclusion Chromatography was used to assay for the effect of the mutation on the rate of dimer disassembly. Contrary to our expectation, the disassembly of dimers of the X-enabled N-cadherin mutant was as slow as seen for wild-type N-cadherin in the apo-state. Thus, the differences in the X-interface residues are not the origin of slow disassembly kinetics of N-cadherin in the apo-state.

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Protein hydrogen exchange: testing current models.

Publication Date: 2012 Apr 27 PMID: 22544567
Authors: Skinner, J. J. - Lim, W. K. - Bedard, S. - Black, B. E. - Englander, S. W.
Journal: Protein Sci

To investigate the determinants of protein hydrogen exchange (HX), HX rates of most of the backbone amide hydrogens of Staphylococcal nuclease were measured by NMR methods. A modified analysis was used to improve accuracy for the faster hydrogens. HX rates of both near surface and well buried hydrogens are spread over more than 7 orders of magnitude. These results were compared with previous hypotheses for HX rate determination. Contrary to a common assumption, proximity to the surface of the native protein does not usually produce fast exchange. The slow HX rates for unprotected surface hydrogens are not well explained by local electrostatic field. The ability of buried hydrogens to exchange is not explained by a solvent penetration mechanism. The exchange rates of structurally protected hydrogens are not well predicted by algorithms that depend only on local interactions or only on transient unfolding reactions. These observations identify some of the present difficulties of HX rate prediction and suggest the need for returning to a detailed hydrogen by hydrogen analysis to examine the bases of structure-rate relationships, as described in the companion paper (Skinner et al. 2012).

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Protein dynamics viewed by hydrogen exchange.

Publication Date: 2012 Apr 27 PMID: 22544544
Authors: Skinner, J. J. - Lim, W. K. - Bedard, S. - Black, B. E. - Englander, S. W.
Journal: Protein Sci

To examine the relationship between protein structural dynamicsand measurable hydrogen exchange (HX) data, the detailed exchange behavior of most of the backbone amide hydrogens of Staphylococcal nucleasewascomparedwith that of their neighbors, with their structural environment, and with other information. Results show that H-bonded hydrogens are protected from exchange, with HX rate effectively zero, even when they are directly adjacent to solvent. The transition to exchange competence requires a dynamic structural excursionthat removes H-bond protection and allows exposure to solvent HX catalyst. The detailed data often make clear the nature of the dynamic excursion required. These range fromwhole molecule unfolding, through smaller cooperative unfolding reactions of secondary structural elements, and down to local fluctuations that involve as little as asingle peptide group or side chain orwater molecule. The particular motion that dominates the exchange of any hydrogen is the one that allows the fastest HX rate. The motionand the rate it produces are determined by surrounding structure and not by nearness to solvent or the strength of the protecting H-bond itself or its acceptor type (main chain, side chain, structurally bound water). Many of these motions occur over time scales that are appropriate for biochemical function.

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Purification, characterization of a CkChn134 protein from Cynanchum komarovii seeds and synergistic effect with CkTLP against Verticillium dahliae.

Publication Date: 2012 Jun PMID: 22532259
Authors: Wang, Q. - Zhang, Y. - Hou, Y. - Wang, P. - Zhou, S. - Ma, X. - Zhang, N.
Journal: Protein Sci

Cynanchum komarovii Al Iljinski is a desert plant that has been used as analgesic, anthelminthic, and antidiarrheal, but also as herbal medicine to treat cholecystitis in people. In this work, an antifungal protein with sequence homology to chitinase was isolated from C. komarovii seeds and named CkChn134. The three-dimensional structure prediction of CkChn134 indicated that the protein has a loop domain formed a thin cleft, which is able to bind molecules and substrates. The protein and CkTLP synergistically inhibited the fungal growth of Verticillium dahliae, Fusarium oxysporum, Rhizoctonia solani, Botrytis cinerea, and Valsa mali in vitro. The full-length cDNA was cloned by RT-PCR and RACE-PCR according to the partial protein sequences obtained by nanoESI-MS/MS. The real-time PCR showed that the transcription level of CkChn134 had a significant increase under the stress of ethylene, NaCl, low temperature, drought, and pathogen infection, which indicates that CkChn134 may play an important role in response to abiotic and biotic stresses. The CkChn134 protein was located in the extracellular space/cell wall by CkChn134::GFP fusion protein in transgenic Arabidopsis. Furthermore, overexpression of CkChn134 significantly enhanced the resistance of transgenic Arabidopsis against V. dahliae. Interestingly, the coexpression of CkChn134 and CkTLP showed substantially greater protection against the fungal pathogen V. dahliae than either transgene alone. The results suggest that the CkChn134 is a good candidate protein or gene, and it had a potential synergistic effect with CkTLP for contributing to the development of disease-resistant crops.

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Structural basis of interleukin-5 dimer recognition by its alpha receptor.

Publication Date: 2012 Jun PMID: 22528658
Authors: Kusano, S. - Kukimoto-Niino, M. - Hino, N. - Ohsawa, N. - Ikutani, M. - Takaki, S. - Sakamoto, K. - Hara-Yokoyama, M. - Shirouzu, M. - Takatsu, K. - Yokoyama, S.
Journal: Protein Sci

Interleukin-5 (IL-5), a major hematopoietin, stimulates eosinophil proliferation, migration, and activation, which have been implicated in the pathogenesis of allergic inflammatory diseases, such as asthma. The specific IL-5 receptor (IL-5R) consists of the IL-5 receptor alpha subunit (IL-5RA) and the common receptor beta subunit (betac). IL-5 binding to IL-5R on target cells induces rapid tyrosine phosphorylation and activation of various cellular proteins, including JAK1/JAK2 and STAT1/STAT5. Here, we report the crystal structure of dimeric IL-5 in complex with the IL-5RA extracellular domains. The structure revealed that IL-5RA sandwiches the IL-5 homodimer by three tandem domains, arranged in a "wrench-like" architecture. This association mode was confirmed for human cells expressing IL-5 and the full-length IL-5RA by applying expanded genetic code technology: protein photo-cross-linking experiments revealed that the two proteins interact with each other in vivo in the same manner as that in the crystal structure. Furthermore, a comparison with the previously reported, partial GM-CSF*GM-CSFRA*betac structure enabled us to propose complete structural models for the IL-5 and GM-CSF receptor complexes, and to identify the residues conferring the cytokine-specificities of IL-5RA and GM-CSFRA.

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The interface of protein structure, protein biophysics, and molecular evolution.

Publication Date: 2012 Jun PMID: 22528593
Authors: Liberles, D. A. - Teichmann, S. A. - Bahar, I. - Bastolla, U. - Bloom, J. - Bornberg-Bauer, E. - Colwell, L. J. - de Koning, A. P. - Dokholyan, N. V. - Echave, J. - Elofsson, A. - Gerloff, D. L. - Goldstein, R. A. - Grahnen, J. A. - Holder, M. T. - Lakner, C. - Lartillot, N. - Lovell, S. C. - Naylor, G. - Perica, T. - Pollock, D. D. - Pupko, T. - Regan, L. - Roger, A. - Rubinstein, N. - Shakhnovich, E. - Sjolander, K. - Sunyaev, S. - Teufel, A. I. - Thorne, J. L. - Thornton, J. W. - Weinreich, D. M. - Whelan, S.
Journal: Protein Sci

Abstract The interface of protein structural biology, protein biophysics, molecular evolution, and molecular population genetics forms the foundations for a mechanistic understanding of many aspects of protein biochemistry. Current efforts in interdisciplinary protein modeling are in their infancy and the state-of-the art of such models is described. Beyond the relationship between amino acid substitution and static protein structure, protein function, and corresponding organismal fitness, other considerations are also discussed. More complex mutational processes such as insertion and deletion and domain rearrangements and even circular permutations should be evaluated. The role of intrinsically disordered proteins is still controversial, but may be increasingly important to consider. Protein geometry and protein dynamics as a deviation from static considerations of protein structure are also important. Protein expression level is known to be a major determinant of evolutionary rate and several considerations including selection at the mRNA level and the role of interaction specificity are discussed. Lastly, the relationship between modeling and needed high-throughput experimental data as well as experimental examination of protein evolution using ancestral sequence resurrection and in vitro biochemistry are presented, towards an aim of ultimately generating better models for biological inference and prediction.

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Active site residues critical for flavin binding and 5,6-dimethylbenzimidazole biosynthesis in the flavin destructase enzyme BluB.

Publication Date: 2012 Jun PMID: 22528544
Authors: Yu, T. Y. - Mok, K. C. - Kennedy, K. J. - Valton, J. - Anderson, K. S. - Walker, G. C. - Taga, M. E.
Journal: Protein Sci

The "flavin destructase" enzyme BluB catalyzes the unprecedented conversion of flavin mononucleotide (FMN) to 5,6-dimethylbenzimidazole (DMB), a component of vitamin B(12) . Because of its unusual chemistry, the mechanism of this transformation has remained elusive. This study reports the identification of 12 mutant forms of BluB that have severely reduced catalytic function, though most retain the ability to bind flavin. The "flavin destructase" BluB is an unusual enzyme that fragments the flavin cofactor FMNH(2) in the presence of oxygen to produce 5,6-dimethylbenzimidazole (DMB), the lower axial ligand of vitamin B(12) (cobalamin). Despite the similarities in sequence and structure between BluB and the nitroreductase and flavin oxidoreductase enzyme families, BluB is the only enzyme known to fragment a flavin isoalloxazine ring. To explore the catalytic residues involved in this unusual reaction, mutants of BluB impaired in DMB biosynthesis were identified in a genetic screen in the bacterium Sinorhizobium meliloti. Of the 16 unique point mutations identified in the screen, the majority were located in conserved residues in the active site or in the unique "lid" domain proposed to shield the active site from solvent. Steady-state enzyme assays of 12 purified mutant proteins showed a significant reduction in DMB synthesis in all of the mutants, with eight completely defective in DMB production. Ten of these mutants have weaker binding affinities for both oxidized and reduced FMN, though only two have a significant effect on complex stability. These results implicate several conserved residues in BluB's unique ability to fragment FMNH(2) and demonstrate the sensitivity of BluB's active site to structural perturbations. This work lays the foundation for mechanistic studies of this enzyme and further advances our understanding of the structure-function relationship of BluB.

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A "Link-Psi" strategy using crosslinking indicates that the folding transition state of ubiquitin is not very malleable.

Publication Date: 2012 Jun PMID: 22528473
Authors: Shandiz, A. T. - Baxa, M. C. - Sosnick, T. R.
Journal: Protein Sci

Using a combined crosslinking-psi analysis strategy, we examine whether the structural content of the transition state of ubiquitin can be altered. A synthetic dichloroacetone crosslink is first introduced across two beta strands. Whether the structural content in the transition state ensemble has shifted towards the region containing the crosslink is probed by remeasuring the psi value at another region (psi identifies the degree to which an inserted bi-Histidine metal ion binding site is formed in the transition state). For sites around the periphery of the obligate transition state nucleus, we find that the resulting changes in psi values are near or at our detection limit, thereby indicating that the structural content of the transition state has not measurably changed upon crosslinking. This work demonstrates the utility of the simultaneous application of crosslinking and psi-analysis for examining potential transition state heterogeneity in globular proteins.

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Structure and dynamics in solution of the stop codon decoding N-terminal domain of the human polypeptide chain release factor eRF1.

Publication Date: 2012 Jun PMID: 22517631
Authors: Polshakov, V. I. - Eliseev, B. D. - Birdsall, B. - Frolova, L. Y.
Journal: Protein Sci

The high-resolution NMR structure of the N-domain of human eRF1, responsible for stop codon recognition, has been determined in solution. The overall fold of the protein is the same as that found in the crystal structure. However, the structures of several loops, including those participating in stop codon decoding, are different. Analysis of the NMR relaxation data reveals that most of the regions with the highest structural discrepancy between the solution and solid states undergo internal motions on the ps-ns and ms time scales. The NMR data show that the N-domain of human eRF1 exists in two conformational states. The distribution of the residues having the largest chemical shift differences between the two forms indicates that helices alpha2 and alpha3, with the NIKS loop between them, can switch their orientation relative to the beta-core of the protein. Such structural plasticity may be essential for stop codon recognition by human eRF1.

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Kinetics of thermally induced heat shock protein 27 and 70 expression by bone marrow-derived mesenchymal stem cells.

Publication Date: 2012 Jun PMID: 22505291
Authors: Moloney, T. C. - Hoban, D. B. - Barry, F. P. - Howard, L. - Dowd, E.
Journal: Protein Sci

Although bone marrow-derived mesenchymal stem cells (MSCs) are an attractive cell therapy candidate, their potential is limited by poor survival following transplantation. Over-expression of anti-apoptotic heat shock proteins using viral vectors can improve the survival of these cells under stressful conditions in vitro and in vivo. It is also possible to induce heat shock protein expression in many cell types by simply exposing them to a transient, nonlethal elevation in temperature. The response profile of MSCs to such a thermal stress has not yet been reported. Therefore, this study sought to determine the kinetics of thermally induced heat shock protein expression by MSCs in vitro. To determine if heat shock protein expression was a function of thermal stress exposure time, MSCs were exposed to 42 degrees C for 15, 30, 45, and 60 min and were harvested 24 h later. To establish the time-course of heat shock protein expression, MSCs were heat shocked for 60 min and harvested 2, 24, 48, 72, 96, and 120 h later. The cells were then analyzed for Hsp27 and Hsp70 expression by Western blot. Densitometric analysis revealed that exposure to a thermal stress induced expression of both Hsp27 and Hsp70 and that the level of expression was dependant on stress exposure time. Following 60 min of heat stress, both Hsp27 and Hsp70 accumulated maximal expression after 48 h with both proteins returning to constitutive expression levels by 120 h. This study demonstrates that heat shock protein expression can be induced in MSCs by a simple thermal stress.

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Structural analysis of a 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase with an N-terminal chorismate mutase-like regulatory domain.

Publication Date: 2012 Jun PMID: 22505283
Authors: Light, S. H. - Halavaty, A. S. - Minasov, G. - Shuvalova, L. - Anderson, W. F.
Journal: Protein Sci

3-Deoxy-D-arabino-heptulosonate 7-phosphate synthase (DAHPS) catalyzes the first step in the biosynthesis of a number of aromatic metabolites. Likely because this reaction is situated at a pivotal biosynthetic gateway, several DAHPS classes distinguished by distinct mechanisms of allosteric regulation have independently evolved. One class of DAHPSs contains a regulatory domain with sequence homology to chorismate mutase-an enzyme further downstream of DAHPS that catalyzes the first committed step in tyrosine/phenylalanine biosynthesis-and is inhibited by chorismate mutase substrate (chorismate) and product (prephenate). Described in this work, structures of the Listeria monocytogenes chorismate/prephenate regulated DAHPS in complex with Mn(2+) and Mn(2+) + phosphoenolpyruvate reveal an unusual quaternary architecture: DAHPS domains assemble as a tetramer, from either side of which chorismate mutase-like (CML) regulatory domains asymmetrically emerge to form a pair of dimers. This domain organization suggests that chorismate/prephenate binding promotes a stable interaction between the discrete regulatory and catalytic domains and supports a mechanism of allosteric inhibition similar to tyrosine/phenylalanine control of a related DAHPS class. We argue that the structural similarity of chorismate mutase enzyme and CML regulatory domain provides a unique opportunity for the design of a multitarget antibacterial.

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Probing the catalytic mechanism of a C-3'-methyltransferase involved in the biosynthesis of D-tetronitrose.

Publication Date: 2012 Jun PMID: 22495991
Authors: Bruender, N. A. - Holden, H. M.
Journal: Protein Sci

D-Tetronitrose is a nitro-containing tetradeoxysugar found attached to the antitumor and antibacterial agent tetrocarcin A. The biosynthesis of this highly unusual sugar in Micromonospora chalcea requires 10 enzymes. The fifth step in the pathway involves the transfer of a methyl group from S-adenosyl-L-methionine (SAM) to the C-3' carbon of dTDP-3-amino-2,3,6-trideoxy-4-keto-D-glucose. The enzyme responsible for this transformation is referred to as TcaB9. It is a monomeric enzyme with a molecular architecture based around three domains. The N-terminal motif contains a binding site for a structural zinc ion. The middle- and C-terminal domains serve to anchor the SAM and dTDP-sugar ligands, respectively, to the protein, and the active site of TcaB9 is wedged between these two regions. For this investigation, the roles of Tyr 76, His 181, Tyr 222, Glu 224, and His 225, which form the active site of TcaB9, were probed by site-directed mutagenesis, kinetic analyses, and X-ray structural studies. In addition, two ternary complexes of the enzyme with bound S-adenosyl-L-homocysteine and either dTDP-3-amino-2,3,6-trideoxy-4-keto-D-glucose or dTDP-3-amino-2,3,6-trideoxy-D-galactose were determined to 1.5 or 1.6 A resolution, respectively. Taken together, these investigations highlight the important role of His 225 in methyl transfer. In addition, the structural data suggest that the methylation reaction occurs via retention of configuration about the C-3' carbon of the sugar.

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SABER: a computational method for identifying active sites for new reactions.

Publication Date: 2012 May PMID: 22492397
Authors: Nosrati, G. R. - Houk, K. N.
Journal: Protein Sci

A software suite, SABER (Selection of Active/Binding sites for Enzyme Redesign), has been developed for the analysis of atomic geometries in protein structures, using a geometric hashing algorithm (Barker and Thornton, Bioinformatics 2003;19:1644-1649). SABER is used to explore the Protein Data Bank (PDB) to locate proteins with a specific 3D arrangement of catalytic groups to identify active sites that might be redesigned to catalyze new reactions. As a proof-of-principle test, SABER was used to identify enzymes that have the same catalytic group arrangement present in o-succinyl benzoate synthase (OSBS). Among the highest-scoring scaffolds identified by the SABER search for enzymes with the same catalytic group arrangement as OSBS were L-Ala D/L-Glu epimerase (AEE) and muconate lactonizing enzyme II (MLE), both of which have been redesigned to become effective OSBS catalysts, demonstrated by experiments. Next, we used SABER to search for naturally existing active sites in the PDB with catalytic groups similar to those present in the designed Kemp elimination enzyme KE07. From over 2000 geometric matches to the KE07 active site, SABER identified 23 matches that corresponded to residues from known active sites. The best of these matches, with a 0.28 A catalytic atom RMSD to KE07, was then redesigned to be compatible with the Kemp elimination using RosettaDesign. We also used SABER to search for potential Kemp eliminases using a theozyme predicted to provide a greater rate acceleration than the active site of KE07, and used Rosetta to create a design based on the proteins identified.

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Is there an en route folding intermediate for Cold shock proteins?

Publication Date: 2012 May PMID: 22467601
Authors: Huang, L. - Shakhnovich, E. I.
Journal: Protein Sci

Cold shock proteins (Csps) play an important role in cold shock response of a diverse number of organisms ranging from bacteria to humans. Numerous studies of the Csp from various species showed that a two-state folding mechanism is conserved and the transition state (TS) appears to be very compact. However, the atomic details of the folding mechanism of Csp remain unclear. This study presents the folding mechanism of Csp in atomic detail using an all-atom Go model-based simulations. Our simulations predict that there may exist an en route intermediate, in which beta strands 1-2-3 are well ordered and the contacts between beta1 and beta4 are almost developed. Such an intermediate might be too unstable to be detected in the previous fluorescence energy transfer experiments. The transition state ensemble has been determined from the P(fold) analysis and the TS appears even more compact than the intermediate state.

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On the unyielding hydrophobic core of villin headpiece.

Publication Date: 2012 May PMID: 22467489
Authors: Brown, J. W. - Farelli, J. D. - McKnight, C. J.
Journal: Protein Sci

Villin headpiece (HP67) is a small, autonomously-folding domain that has become a model system for understanding the fundamental tenets governing protein folding. In this communication, we explore the role that Leu61 plays in the structure and stability of the construct. Deletion of Leu61 results in a completely unfolded protein that cannot be expressed in Escherichia coli. Omission of only the aliphatic leucine side chain (HP67 L61G) perturbed neither the backbone conformation nor the orientation of local hydrophobic side chains. As a result, a large, solvent-exposed hydrophobic pocket, a negative replica of the leucine side-chain, was created on the surface. The loss of the hydrophobic interface between leucine 61 and the hydrophobic pocket destabilized the construct by ~3.3 kcal/mol. Insertion of a single glycine residue immediately before Leu61 (HP67 L61[GL]) was also highly destabilizing and had the effect of altering the backbone conformation (alpha-helix to pi-helix) in order to precisely preserve the wild-type position and conformation of all hydrophobic residues, including Leu61. In addition to demonstrating that the hydrophobic side-chain of Leu61 is critically important for the stability of villin headpiece, our results are consistent with the notion that the precise interactions present within the hydrophobic core, rather than the hydrogen bonds that define the secondary structure, specify a protein's fold.

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Biodegradation of elastin-like polypeptide nanoparticles.

Publication Date: 2012 Jun PMID: 22434766
Authors: Shah, M. - Hsueh, P. Y. - Sun, G. - Chang, H. Y. - Janib, S. M. - Mackay, J. A.
Journal: Protein Sci

Protein polymers are repetitive polypeptides produced by ribosomal biosynthetic pathways; furthermore, they offer emerging opportunities in drug and biopharmaceutical delivery. As for any polymer, biodegradation is one of the most important determinants affecting how a protein polymer can be utilized in the body. This study was designed to characterize the proteolytic biodegradation for a library of protein polymers derived from the human tropoelastin, the Elastin-like polypeptides (ELPs). ELPs are of particular interest for controlled drug delivery because they reversibly transition from soluble to insoluble above an inverse phase transition temperature (T(t) ). More recently, ELP block copolymers have been developed that can assemble into micelles; however, it remains unclear if proteases can act on these ELP nanoparticles. For the first time, we demonstrate that ELP nanoparticles can be degraded by two model proteases and that comparable proteolysis occurs after cell uptake into a transformed culture of murine hepatocytes. Both elastase and collagenase endopeptidases can proteolytically degrade soluble ELPs. To our surprise, the ELP phase transition was protective against collagenase but not to elastase activity. These findings enhance our ability to predict how ELPs will biodegrade in different physiological microenvironments and are essential to develop protein polymers into biopharmaceuticals.

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Caspases cleave and inhibit the microRNA processing protein DiGeorge Critical Region 8.

Publication Date: 2012 Jun PMID: 22434730
Authors: Gong, M. - Chen, Y. - Senturia, R. - Ulgherait, M. - Faller, M. - Guo, F.
Journal: Protein Sci

DGCR8 (DiGeorge Critical Region 8) is an essential microRNA (miRNA) processing protein that recognizes primary transcripts of miRNAs (pri-miRNAs) and triggers their cleavage by the Drosha nuclease. We previously found that Fe(III) heme binds and activates DGCR8. Here we report that in HeLa cells, DGCR8 undergoes two proteolytic events that produce two C-terminal fragments called DGCR8(C1) and DGCR8(C2) , respectively. DGCR8(C2) accumulates during apoptosis and is generated through cleavage by a caspase. The caspase cleavage site is located in the central loop of the heme-binding domain. Cleavage of DGCR8 by caspase-3 in vitro results in loss of the otherwise tightly bound Fe(III) heme cofactor, dissociation of the N- and C-terminal proteolytic fragments, and inhibition of the pri-miRNA processing activity. These results reveal an intrinsic mechanism in the DGCR8 protein that seems to have evolved for regulating miRNA processing via association with Fe(III) heme and proteolytic cleavage by caspases. Decreased expression of miRNAs has been observed in apoptotic cells, and this change was attributed to caspase-mediated cleavage of a down-stream miRNA processing nuclease Dicer. We suggest that both the Drosha and Dicer cleavage steps of the miRNA maturation pathway may be inhibited in apoptosis and other biological processes where caspases are activated.

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The protein gp74 from the bacteriophage HK97 functions as a HNH endonuclease.

Publication Date: 2012 Jun PMID: 22434504
Authors: Moodley, S. - Maxwell, K. L. - Kanelis, V.
Journal: Protein Sci

The last gene in the genome of the bacteriophage HK97 encodes the protein gp74. We present data in this article that demonstrates, for the first time, that gp74 possesses HNH endonuclease activity. HNH endonucleases are small DNA binding and digestion proteins characterized by two His residues and an Asn residue. We demonstrate that gp74 cleaves lambda phage DNA at multiple sites and that gp74 requires divalent metals for its endonuclease activity. We also present intrinsic tryptophan fluorescence data that show direct binding of Ni(2+) to gp74. The activity of gp74 in the presence of Ni(2+) is significantly decreased below neutral pH, suggesting the presence of one or more His residues in metal binding and/or DNA digestion. Surprisingly, this pH-dependence of activity is not seen with Zn(2+) , suggesting a different mode of binding of Zn(2+) and Ni(2+) . This difference in activity may result from binding of a second Zn(2+) ion by a putative zinc finger in gp74 in addition to binding of a Zn(2+) ion by the HNH motif. These studies define the biochemical function of gp74 as an HNH endonuclease and provide a platform for determining the role of gp74 in life cycle of the bacteriophage HK97.

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Characterization of deamidation of barstar using electrospray ionization quadrupole time-of-flight mass spectrometry, which stabilizes an equilibrium unfolding intermediate.

Publication Date: 2012 May PMID: 22431291
Authors: Jha, S. K. - Deepalakshmi, P. D. - Udgaonkar, J. B.
Journal: Protein Sci

Deamidation of asparaginyl residues is a common posttranslational modification in proteins and has been studied extensively because of its important biological effects, such as those on enzymatic activity, protein folding, and proteolytic degradation. However, characterization of the sites of deamidation of a protein has been a difficult analytical problem. In this study, mass spectrometry has been used as an analytical tool to characterize the deamidation of barstar, an RNAse inhibitor. Upon incubation of the protein at alkaline pH for 5 h, intact mass analysis of barstar, using electrospray ionization quadrupole time-of-flight mass spectrometry (ESI QToF MS), indicated an increase in the mass of +2 Da, suggesting possible deamidation of the protein. The sites of deamidation have been identified using the conventional bottom-up approach using a capillary liquid chromatography connected on line to an ESI QToF mass spectrometer and top down approach by direct infusion of the intact protein and fragmenting inside MS. These chemical modifications are shown to lead to stabilization of an unfolding intermediate, which can be observed in equilibrium unfolding studies.

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The extension peptide of plant ferritin from sea lettuce contributes to shell stability and surface hydrophobicity.

Publication Date: 2012 Jun PMID: 22419613
Authors: Masuda, T. - Morimoto, S. - Mikami, B. - Toyohara, H.
Journal: Protein Sci

Plant ferritins have some unique structural and functional features. Most of these features can be related to the plant-specific "extension peptide" (EP), which exists in the N-terminus of the mature region of a plant ferritin. Recent crystallographic analysis of a plant ferritin revealed the structure of the EP, however, two points remain unclear: (i) whether the structures of well-conserved EP of plant ferritins are common in all plants, and (ii) whether the EP truly contributes to the shell stability of the plant ferritin oligomer. To clarify these matters, we have cloned a green-plant-type ferritin cDNA from a green alga, Ulva pertusa, and investigated its crystal structure. Ulva pertusa ferritin (UpFER) has a plant-ferritin-specific extension peptide composed of 28 amino acid residues. In the crystal structure of UpFER, the EP lay on and interacted with the neighboring threefold symmetry-related subunit. The amino acid residues involved in the interaction were very highly conserved among plant ferritins. The EPs masked the hydrophobic pockets on the ferritin shell surface by lying on them, and this made the ferritin oligomer more hydrophilic. Furthermore, differential scanning calorimetric analysis of the native and its EP-deletion mutant suggested that the EP contributed to the thermal stability of the plant ferritin shell. Thus, the shell stability and surface hydrophobicity of plant ferritin were controlled by the presence or absence of the plant-ferritin-specific EP. This regulation can account for those processes such as shell stability, degradation, and association of plant ferritin, which are significantly related to iron utilization in plants.

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Glu121-Lys319 salt bridge between catalytic and N-terminal domains is pivotal for the activity and stability of Escherichia coli aminopeptidase N.

Publication Date: 2012 May PMID: 22411732
Authors: Gumpena, R. - Kishor, C. - Ganji, R. J. - Jain, N. - Addlagatta, A.
Journal: Protein Sci

Escherichia coli aminopeptidase N (ePepN) belongs to the gluzincin family of M1 class metalloproteases that share a common primary structure with consensus zinc binding motif (HEXXH-(X18)-E) and an exopeptidase motif (GXMEN) in the active site. There is one amino acid, E121 in Domain I that blocks the extended active site grove of the thermolysin like catalytic domain (Domain II) limiting the substrate to S1 pocket. E121 forms a part of the S1 pocket, while making critical contact with the amino-terminus of the substrate. In addition, the carboxylate of E121 forms a salt bridge with K319 in Domain II. Both these residues are absolutely conserved in ePepN homologs. Analogous Glu-Asn pair in tricon interacting factor F3 (F3) and Gln-Asn pair in human leukotriene A(4) hydrolase (LTA(4) H) are also conserved in respective homologs. Mutation of either of these residues individually or together substantially reduced or entirely eliminated enzymatic activity. In addition, thermal denaturation studies suggest that the mutation at K319 destabilizes the protein as much as by 3.7 degrees C, while E121 mutants were insensitive. Crystal structure of E121Q mutant reveals that the enzyme is inactive due to the reduced S1 subsite volume. Together, data presented here suggests that ePepN, F3, and LTA(4) H homologs adopted a divergent evolution that includes E121-K319 or its analogous pairs, and these cannot be interchanged.

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Robust design and optimization of retroaldol enzymes.

Publication Date: 2012 May PMID: 22407837
Authors: Althoff, E. A. - Wang, L. - Jiang, L. - Giger, L. - Lassila, J. K. - Wang, Z. - Smith, M. - Hari, S. - Kast, P. - Herschlag, D. - Hilvert, D. - Baker, D.
Journal: Protein Sci

Enzyme catalysts of a retroaldol reaction have been generated by computational design using a motif that combines a lysine in a nonpolar environment with water-mediated stabilization of the carbinolamine hydroxyl and beta-hydroxyl groups. Here, we show that the design process is robust and repeatable, with 33 new active designs constructed on 13 different protein scaffold backbones. The initial activities are not high but are increased through site-directed mutagenesis and laboratory evolution. Mutational data highlight areas for improvement in design. Different designed catalysts give different borohydride-reduced reaction intermediates, suggesting a distribution of properties of the designed enzymes that may be further explored and exploited.

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Structural and enzymatic characterization of BacD, an L-amino acid dipeptide ligase from Bacillus subtilis.

Publication Date: 2012 May PMID: 22407814
Authors: Shomura, Y. - Hinokuchi, E. - Ikeda, H. - Senoo, A. - Takahashi, Y. - Saito, J. - Komori, H. - Shibata, N. - Yonetani, Y. - Higuchi, Y.
Journal: Protein Sci

BacD is an ATP-dependent dipeptide ligase responsible for the biosynthesis of L-alanyl-L-anticapsin, a precursor of an antibiotic produced by Bacillus spp. In contrast to the well-studied and phylogenetically related D-alanine: D-alanine ligase (Ddl), BacD synthesizes dipeptides using L-amino acids as substrates and has a low substrate specificity in vitro. The enzyme is of great interest because of its potential application in industrial protein engineering for the environmentally friendly biological production of useful peptide compounds, such as physiologically active peptides, artificial sweeteners and antibiotics, but the determinants of its substrate specificity and its catalytic mechanism have not yet been established due to a lack of structural information. In this study, we report the crystal structure of BacD in complex with ADP and an intermediate analog, phosphorylated phosphinate L-alanyl-L-phenylalanine, refined to 2.5-A resolution. The complex structure reveals that ADP and two magnesium ions bind in a manner similar to that of Ddl. However, the dipeptide orientation is reversed, and, concomitantly, the entrance to the amino acid binding cavity differs in position. Enzymatic characterization of two mutants, Y265F and S185A, demonstrates that these conserved residues are not catalytic residues at least in the reaction where L-phenylalanine is used as a substrate. On the basis of the biochemical and the structural data, we propose a reaction scheme and a catalytic mechanism for BacD.

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N-Terminal acetylation is critical for forming alpha-helical oligomer of alpha-synuclein.

Publication Date: 2012 May PMID: 22407793
Authors: Trexler, A. J. - Rhoades, E.
Journal: Protein Sci

The aggregation of the protein alpha-synuclein (AS) is critical to the pathogenesis of Parkinson's disease. Although generally described as an unstructured monomer, recent evidence suggests that the native form of AS may be an alpha-helical tetramer which resists aggregation. Here, we show that N-terminal acetylation in combination with a mild purification protocol results in an oligomeric form of AS with partial alpha-helical structure. N-terminal acetylation of AS could have important implications for both the native and pathological structures and functions of AS. Through our demonstration of a recombinant expression system, our results represent an important step toward biochemical and biophysical characterization of this potentially important form of AS.

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Formyl-coenzyme A (CoA):oxalate CoA-transferase from the acidophile Acetobacter aceti has a distinctive electrostatic surface and inherent acid stability.

Publication Date: 2012 May PMID: 22374910
Authors: Mullins, E. A. - Starks, C. M. - Francois, J. A. - Sael, L. - Kihara, D. - Kappock, T. J.
Journal: Protein Sci

Bacterial formyl-CoA:oxalate CoA-transferase (FCOCT) and oxalyl-CoA decarboxylase work in tandem to perform a proton-consuming decarboxylation that has been suggested to have a role in generalized acid resistance. FCOCT is the product of uctB in the acidophilic acetic acid bacterium Acetobacter aceti. As expected for an acid-resistance factor, UctB remains folded at the low pH values encountered in the A. aceti cytoplasm. A comparison of crystal structures of FCOCTs and related proteins revealed few features in UctB that would distinguish it from nonacidophilic proteins and thereby account for its acid stability properties, other than a strikingly featureless electrostatic surface. The apparently neutral surface is a result of a "speckled" charge decoration, in which charged surface residues are surrounded by compensating charges but do not form salt bridges. A quantitative comparison among orthologs identified a pattern of residue substitution in UctB that may be a consequence of selection for protein stability by constant exposure to acetic acid. We suggest that this surface charge pattern, which is a distinctive feature of A. aceti proteins, creates a stabilizing electrostatic network without stiffening the protein or compromising protein-solvent interactions.

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Ratcheting up protein translocation with anthrax toxin.

Publication Date: 2012 May PMID: 22374876
Authors: Feld, G. K. - Brown, M. J. - Krantz, B. A.
Journal: Protein Sci

Energy-consuming nanomachines catalyze the directed movement of biopolymers in the cell. They are found both dissolved in the aqueous cytosol as well as embedded in lipid bilayers. Inquiries into the molecular mechanism of nanomachine-catalyzed biopolymer transport have revealed that these machines are equipped with molecular parts, including adjustable clamps, levers, and adaptors, which interact favorably with substrate polypeptides. Biological nanomachines that catalyze protein transport, known as translocases, often require that their substrate proteins unfold before translocation. An unstructured protein chain is likely entropically challenging to bind, push, or pull in a directional manner, especially in a way that produces an unfolding force. A number of ingenious solutions to this problem are now evident in the anthrax toxin system, a model used to study protein translocation. Here we highlight molecular ratchets and current research on anthrax toxin translocation. A picture is emerging of proton-gradient-driven anthrax toxin translocation, and its associated ratchet mechanism likely applies broadly to other systems. We suggest a cyclical thermodynamic order-to-disorder mechanism (akin to a heat-engine cycle) is central to underlying protein translocation: peptide substrates nonspecifically bind to molecular clamps, which possess adjustable affinities; polypeptide substrates compress into helical structures; these clamps undergo proton-gated switching; and the substrate subsequently expands regaining its unfolded state conformational entropy upon translocation.

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Structural basis of RNA binding by leucine zipper GCN4.

Publication Date: 2012 May PMID: 22374868
Authors: Nikolaev, Y. - Pervushin, K.
Journal: Protein Sci

Recently, we showed that leucine zipper (LZ) motifs of basic leucine zipper (bZIP) transcription factors GCN4 and c-Jun are capable of catalyzing degradation of RNA (Nikolaev et al., PLoS ONE 2010; 5:e10765). This observation is intriguing given the tight regulation of RNA turnover control and the antiquity of bZIP transcription factors. To support further mechanistic studies, herein, we elucidated RNA binding interface of the GCN4 leucine zipper motif from yeast. Solution NMR experiments showed that the LZ-RNA interaction interface is located in the first two heptads of LZ moiety, and that only the dimeric (coiled coil) LZ conformation is capable of binding RNA. Site-directed mutagenesis of the LZ-GCN4 RNA binding interface showed that substrate binding is facilitated by lysine and arginine side chains, and that at least one nucleophilic residue is located in proximity to the RNA phosphate backbone. Further studies in the context of full-length bZIP factors are envisaged to address the biological relevance of LZ RNase activity.

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Acyl carrier protein structural classification and normal mode analysis.

Publication Date: 2012 May PMID: 22374859
Authors: Cantu, D. C. - Forrester, M. J. - Charov, K. - Reilly, P. J.
Journal: Protein Sci

All acyl carrier protein primary and tertiary structures were gathered into the ThYme database. They are classified into 16 families by amino acid sequence similarity, with members of the different families having sequences with statistically highly significant differences. These classifications are supported by tertiary structure superposition analysis. Tertiary structures from a number of families are very similar, suggesting that these families may come from a single distant ancestor. Normal vibrational mode analysis was conducted on experimentally determined freestanding structures, showing greater fluctuations at chain termini and loops than in most helices. Their modes overlap more so within families than between different families. The tertiary structures of three acyl carrier protein families that lacked any known structures were predicted as well.

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Inositol phosphate-induced stabilization of inositol 1,3,4,5,6-pentakisphosphate 2-kinase and its role in substrate specificity.

Publication Date: 2012 May PMID: 22362712
Authors: Gosein, V. - Leung, T. F. - Krajden, O. - Miller, G. J.
Journal: Protein Sci

Inositol phosphate kinases (IPKs) sequentially phosphorylate inositol phosphates (IPs) on their inositol rings to yield an array of signaling molecules. IPKs must possess the ability to recognize their physiological substrates from among a pool of over 30 cellular IPs that differ in numbers and positions of phosphates. Crystal structures from IPK subfamilies have revealed structural determinants for IP discrimination, which vary considerably between IPKs. However, recent structures of inositol 1,3,4,5,6-pentakisphosphate 2-kinase (IPK1) did not reveal how IPK1 selectively recognizes its physiological substrate, IP5, while excluding others. Here, we report that limited proteolysis has revealed the presence of multiple conformational states in the IPK1 catalytic cycle, with notable protection from protease only in the presence of IP. Further, a 3.1-A crystal structure of IPK1 bound to ADP in the absence of IP revealed decreased order in residues 110-140 within the N-lobe of the kinase compared with structures in which IP is bound. Using this solution and crystallographic data, we propose a model for recognition of IP substrate by IPK1 wherein phosphate groups at the 4-, 5-, and 6-positions are recognized initially by the C-lobe with subsequent interaction of the 1-position phosphate by Arg130 that stabilizes this residue and the N-lobe. This model explains how IPK1 can be highly specific for a single IP substrate by linking its interactions with substrate phosphate groups to the stabilization of the N- and C-lobes and kinase activation.

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Glycine to glutamic acid misincorporation observed in a recombinant protein expressed by Escherichia coli cells.

Publication Date: 2012 May PMID: 22362707
Authors: Huang, Y. - O'Mara, B. - Conover, M. - Ludwig, R. - Fu, J. - Tao, L. - Li, Z. J. - Rieble, S. - Grace, M. J. - Russell, R. J.
Journal: Protein Sci

A novel amino acid misincorporation, in which the intended glycine (Gly) residues were replaced by a glutamic acid (Glu), was observed in a recombinant protein expressed by Escherichia coli. The misincorporation was identified by peptide mapping and liquid chromatography-tandem mass spectrometric analysis on proteolyzed peptides of the protein and verified using the corresponding synthetic peptides containing the misincorporated residues. Analysis of the distribution of the misincorporated residues and their codon usage shows strong correlation between this misincorporation and the use of rarely used codon within the E. coli expression system. Results in this study suggest that the usage of the rare codon GGA has resulted in a Glu for Gly misincorporation.

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