Proteins

Thermodynamic basis of selectivity in guide-target-mismatched RNA interference.

Publication Date: 2012 Jan 9 PMID: 22275138
Authors: Joseph, T. T. - Osman, R.
Journal: Proteins

Silencing in RNAi is strongly affected by guide-strand/target-mRNA mismatches. Target nucleation is thought to occur at positions 2-8 of the guide ("seed region"); successful hybridization in this region is the primary determinant of target binding affinity and hence target cleavage. To define a molecular basis for the target sequence selectivity in RNAi, we studied all possible distinct single mismatches in seven positions of the seed region - a total of 21 substitutions. We report results from soft-core thermodynamic integration simulations to determine changes in target binding free energies to Argonaute due to single mismatches in the guide strand, which arise during binding of an imperfectly matched target mRNA. In agreement with experiment, most mismatches impair target binding, consistent with a prominent role for binding affinity changes in RNAi sequence selectivity. Individual Argonaute residues located near the mismatched base pair are found to contribute significantly to binding affinity changes. We also employ this methodology to analyze the mismatch-dependent free energy changes for dissociation of a DNA*RNA hybrid from Argonaute, as a model for the escape of miRNAs from the silencing pathway. Several mismatched sequences of the miRNA have increased affinity to Argonaute, implying that some mismatches may reduce the probability for escape. Furthermore, calculations of base-substitution-dependent free energy changes for binding ssDNA reveal mild sequence sensitivity as expected for guide strand binding to Argonaute. Our findings give a thermodynamic basis for RNAi target sequence selectivity and suggest that miRNA mismatches may increase silencing effectiveness and thus could be evolutionarily advantageous. Proteins 2012. (c) 2012 Wiley-Liss, Inc.

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Crystal structure and fluorescence studies reveal the role of helical dimeric interface of staphylococcal FabG1 in positive cooperativity for NADPH.

Publication Date: 2012 Jan 9 PMID: 22275129
Authors: Dutta, D. - Bhattacharyya, S. - Das, A. K.
Journal: Proteins

Crystal structure of Staphylococcal beta-ketoacyl-ACP reductase 1 (SaFabG1) complexed with NADPH is determined at 2.5 A resolution. The enzyme is essential in FAS-II pathway and utilizes NADPH to reduce beta-ketoacyl-ACP to (S)-beta-hydroxyacyl-ACP. Unlike the tetrameric FabGs, dimeric SaFabG1 shows positive homotropic cooperativity towards NADPH. Analysis of FabG:NADPH binary crystal structure endorses that NADPH interacts directly with the helices alpha4 and alpha5 those are present on a dimerization interface. A steady shift in tryptophan (of alpha4 helix) emission peak upon steady increment of NADPH concentration reveals that the dimeric interface is formed by alpha4-alpha4' and alpha5-alpha5' helices. This dimeric interface imparts positive homotropic cooperativity towards NADPH. PEG, a substrate mimicking molecule is also found near the active site of the enzyme. Proteins 2012. (c) 2012 Wiley-Liss, Inc.

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A synthetic hexapeptide designed to resemble a proteinaceous p-loop nest is shown to bind inorganic phosphate.

Publication Date: 2012 Jan 18 PMID: 22275093
Authors: Bianchi, A. - Giorgi, C. - Ruzza, P. - Toniolo, C. - Milner-White, E. J.
Journal: Proteins

The hexapeptide Ser-Gly-Ala-Gly-Lys-Thr has been synthesized and characterized. It was designed as a minimal soluble peptide that would be likely to have the phosphate-binding properties observed in the P-loops of proteins that bind the beta-phosphate of GTP or ATP. The beta-phosphate in such proteins is bound by a combination of the side chain epsilon-amino group of the lysine residue plus the concavity formed by successive main chain peptide NH groups called a nest, which is favored by the glycines. The hexapeptide is shown to bind HPO(4) (2-) strongly at neutral pH. The affinities of the various ionized species of phosphate and hexapeptide are analyzed, showing that they increase with pH. It is likely the main chain NH groups of the hexapeptide bind phosphate in much the same way as the corresponding P-loop atoms bind the phosphate ligand in proteins. Most proteinaceous P-loops are situated at the N-termini of alpha-helices and this observation has frequently been considered a key aspect of these binding sites. Such a hexapeptide in isolation seems unlikely to form an alpha-helix, an expectation in accord with the CD spectra examined; this suggests that being at the N-terminus of an a-helix is not essential for phosphate binding. An unexpected finding about the hexapeptide-HPO(4) (2-) complex is that the side chain epsilon-amino group of the lysine occurs in its deprotonated form, which appears to bind HPO(4) (2-) via an N...H-O-P hydrogen bond. (c) 2012 Wiley-Liss, Inc.

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Molecular modeling of hair keratin/peptide complex: Using MM-PBSA calculations to describe experimental binding results.

Publication Date: 2012 Jan 18 PMID: 22275089
Authors: Azoia, N. G. - Fernandes, M. M. - Micaelo, N. M. - Soares, C. M. - Cavaco-Paulo, A.
Journal: Proteins

Molecular dynamics simulations of a keratin/peptide complex have been conducted to predict the binding affinity of four different peptides towards human hair. Free energy calculations on the peptides' interaction with the keratin model demonstrated that electrostatic interactions are believed to be the main driving force stabilizing the complex. The MM-PBSA methodology used for the free energy calculations demonstrated that the dielectric constant in the protein's interior plays a major role in the free energy calculations, and the only way to obtain accordance between the free energy calculations and the experimental binding results was to use the average dielectric constant. (c) 2012 Wiley-Liss, Inc.

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Towards a systems level view of the ECM and related proteins: A framework for the systematic definition and analysis of biological systems.

Publication Date: 2012 Jan 17 PMID: 22275077
Authors: Cromar, G. L. - Xiong, X. - Chautard, E. - Ricard-Blum, S. - Parkinson, J.
Journal: Proteins

Advances in high throughput 'omic technologies are starting to provide unprecedented insights into how components of biological systems are organized and interact. Key to exploiting these datasets is the definition of the components that comprise the system of interest. While a variety of knowledgebases exist that capture such information, a major challenge is determining how these resources may be best utilized. Here we present a systematic curation strategy to define a systems-level view of the human extracellular matrix (ECM) - a three-dimensional meshwork of proteins and polysaccharides that impart structure and mechanical stability to tissues. Employing our curation strategy we define a set of 357 proteins that represent core components of the ECM, together with an additional 524 genes that mediate related functional roles, and construct a map of their physical interactions. Topological properties help identify modules of functionally related proteins, including those involved in cell adhesion, bone formation and blood clotting. Due to its major role in cell adhesion, proliferation and morphogenesis, defects in the ECM have been implicated in cancer, atherosclerosis, asthma, fibrosis and arthritis. We use MeSH annotations to identify modules enriched for specific disease terms that aid to strengthen existing as well as predict novel gene-disease associations. Mapping expression and conservation data onto the network reveal modules evolved in parallel to convey tissue specific functionality on otherwise broadly expressed units. In addition to demonstrating an effective workflow for defining biological systems, this study crystallizes our current knowledge surrounding the organization of the ECM. Proteins 2012. (c) 2012 Wiley-Liss, Inc.

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Do crystal structures obviate the need for theoretical models of GPCRs for structure based virtual screening.

Publication Date: 2012 Jan 17 PMID: 22275072
Authors: Tang, H. - Wang, X. S. - Hsieh, J. H. - Tropsha, A.
Journal: Proteins

Recent highly expected structural characterizations of agonist-bound and antagonist-bound beta-2 adrenoreceptor (beta2AR) by X-ray crystallography have been widely regarded as critical advances to enable more effective structure-based discovery of GPCRs ligands. It appears that this very important development may have undermined many previous efforts to develop 3D theoretical models of GPCRs. To address this question directly we have compared several historical beta2AR models versus the inactive state and nanobody-stabilized active state of beta2AR crystal structures in terms of their structural similarity and effectiveness of use in virtual screening for beta2AR specific agonists and antagonists. Theoretical models, incluing both homology and de novo types, were collected from five different groups who have published extensively in the field of GPCRs modeling; all models were built before X-ray structures became available. In general, beta2AR theoretical models differ significantly from the crystal structure in terms of TMH definition and the global packing. Nevertheless, surprisingly, several models afforded hit rates resulting from virtual screening of large chemical library enriched by known beta2AR ligands that exceeded those using X-ray structures; the hit rates were particularly higher for agonists. Furthemore, the screening performance of models is associated with local structural quality such as the RMSDs for binding pocket residues and the ability to capture accurately most if not all critical protein/ligand interactions. These results suggest that carefully built models of GPCRs could capture critical chemical and structural features of the binding pocket thus may be even more useful for practical structure-based drug discovery than X-ray structures. Proteins 2012. (c) 2012 Wiley-Liss, Inc.

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The binding affinities of proteins interacting with the PDZ domain of PICK1.

Publication Date: 2012 Jan 17 PMID: 22275068
Authors: Bolia, A. - Gerek, Z. N. - Keskin, O. - Ozkan, S. B. - Dev, K. K.
Journal: Proteins

Protein interacting with C kinase (PICK1) is well conserved throughout evolution and plays a critical role in synaptic plasticity by regulating the trafficking and posttranslational modification of its interacting proteins. PICK1 contains a single PSD95/DlgA/Zo1 (PDZ) proteinprotein interaction domain, which is promiscuous and shown to interact with over 60 proteins, most of which play roles in neuronal function. Several reports have suggested the role of PICK1 in disorders such as epilepsy, pain, brain trauma and stroke, drug abuse and dependence, schizophrenia and psychosis. Importantly, lead compounds that block PICK1 interactions are also now becoming available. Here, a new modeling approach was developed to investigate binding affinities of PDZ interactions. Using these methods, the binding affinities of all major PICK1 interacting proteins are reported and the effects of PICK1 mutations on these interactions are described. These modeling methods have important implications in defining the binding properties of proteins interacting with PICK1 as well as the general structural requirements of PDZ interactions. The study also provides modeling methods to support in the drug design of ligands for PDZ domains, which may further aid in development of the family of PDZ domains as a drug target. Proteins 2012. (c) 2012 Wiley-Liss, Inc.

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Thermodynamic stability, unfolding kinetics, and aggregation of the N-terminal actin binding domains of utrophin and dystrophin.

Publication Date: 2012 Jan 9 PMID: 22275054
Authors: Singh, S. M. - Molas, J. F. - Kongari, N. - Bandi, S. - Armstrong, G. S. - Winder, S. J. - Mallela, K. M.
Journal: Proteins

Muscular dystrophy (MD) is the most common genetic lethal disorder in children. Mutations in dystrophin trigger the most common form of MD, Duchenne and its allelic variant Becker MD. Utrophin is the closest homologue and has been shown to compensate for the loss of dystrophin in human disease animal models. However, the structural and functional similarities and differences between utrophin and dystrophin are less understood. Both proteins interact with actin through their N-terminal actin-binding domain (N-ABD). In this study, we examined the thermodynamic stability and aggregation of utrophin N-ABD and compared with that of dystrophin. Our results show that utrophin N-ABD has spectroscopic properties similar to dystrophin N-ABD. However, utrophin N-ABD has decreased denaturant and thermal stability, unfolds faster, and is correspondingly more susceptible to proteolysis, which might account for its decreased in-vivo half-life compared to dystrophin. In addition, utrophin N-ABD aggregates to a lesser extent compared with dystrophin N-ABD, contrary to the general behavior of proteins in which decreased stability enhances protein aggregation. Despite these differences in stability and aggregation, both proteins exhibit deleterious effects of mutations. When utrophin N-ABD mutations analogous in position to the dystrophin disease-causing mutations were generated, they behaved similarly to dystrophin mutants in terms of decreased stability and the formation of cross-beta aggregates, indicating a possible role for utrophin mutations in disease mechanisms. Proteins 2012. (c) 2012 Wiley-Liss, Inc.

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Signatures of the ATP binding pocket as a basis for structural classification of the serine/threonine protein kinases of gram-positive bacteria.

Publication Date: 2012 Jan 9 PMID: 22275035
Authors: Zakharevich, N. V. - Osolodkin, D. I. - Artamonova, I. I. - Palyulin, V. A. - Zefirov, N. S. - Danilenko, V. N.
Journal: Proteins

Eukaryotic-like serine/threonine protein kinases (ESTPKs) are widely spread throughout the bacterial genomes. These enzymes can be potential targets of new anti-bacterial drugs useful for treatment of socially important diseases such as tuberculosis. In this study, ESTPKs of pathogenic, probiotic and antibiotic-producing gram-positive bacteria were classified according to the physico-chemical properties of amino acid residues in the ATP binding site of the enzyme. Nine residues were identified that line the surface of the adenine binding pocket, and ESTPKs were classified based on these signatures. Twenty groups were discovered, five of them containing >10 representatives. The two most abundant groups contained >150 protein kinases that belong to the various branches of the phylogenetic tree, whereas certain groups are genus- or even species-specific. Homology modeling of the typical representatives of each group revealed that the classification is reliable and the differences between the protein kinase ATP binding pockets predicted based on their signatures are apparent in their structure. The classification is expected to be useful for selection of targets for new anti-infective drugs. Proteins 2012. (c) 2012 Wiley-Liss, Inc.

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Signaling pathway of a photoactivable Rac1-GTPase in the early stages.

Publication Date: 2012 Jan 9 PMID: 22275005
Authors: Peter, E. - Dick, B. - Baeurley, S. A.
Journal: Proteins

In modern life- and medical-sciences major efforts are currently concentrated on creating arti-ficial photoenzymes, consisting of light-oxygen-voltage-sensitive (LOV) domains fused to a target enzyme. Such protein constructs possess great potential for controlling the cell metabolism as well as gene function upon light stimulus. This has recently been impressively demonstrated by de- signing a novel artificial fusion protein, connecting the AsLOV2-Jalpha-photosensor from Avena sativa with the Rac1-GTPase (AsLOV2-Jalpha-Rac1), and by using it, to control the motility of cancer cells from the HeLa-line. Although tremendous progress has been achieved on the generation of such protein constructs, a detailed understanding of their signaling pathway after photoexcitation is still in its infancy. Here, we show through computer simulations of the AsLOV2-Jalpha-Rac1-photoenzyme that the early processes after formation of the Cys450-FMN-adduct involve the breakage of a H- bond between the carbonyl oxygen FMN-C4=O and the amino group of Gln513, followed by a rotational re-orientation of its sidechain. This initial event is followed by successive events includ- ing beta-sheet tightening and transmission of torsional stress along the Ibeta-sheet, which leads to the disruption of the Jalpha-helix from the N-terminal end. Finally, this process triggers the detachment of the AsLOV2-Jalpha-photosensor from the Rac1-GTPase, ultimately enabling the activation of Rac1 via binding of the effector protein PAK1. Proteins 2012. (c) 2012 Wiley-Liss, Inc.

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A residue in helical conformation in the native state adopts a beta- strand conformation in the folding transition state despite its high and canonical Phi-value.

Publication Date: 2012 Jan 9 PMID: 22274997
Authors: Zarrine-Afsar, A. - Dahesh, S. - Davidson, A. R.
Journal: Proteins

Delineating structures of the transition states in protein folding reactions has provided great insight into the mechanisms by which proteins fold. The most common method for obtaining this information is Phi-value analysis, which is carried out by measuring the changes in the folding and unfolding rates caused by single amino acid substitutions at various positions within a given protein. Canonical Phi-values range between 0 and 1, and residues displaying high values within this range are interpreted to be important in stabilizing the transition state structure, and to elicit this stabilization through native-like interactions. Although very successful in defining the general features of transition state structures, Phi-value analysis can be confounded when non-native interactions stabilize this state. In addition, direct information on backbone conformation within the transition state is not provided. In the work described here, we have investigated structure formation at a conserved beta-bulge (with helical conformation) in the Fyn SH3 domain by characterizing the effects of substituting all natural amino acids at one position within this structural motif. By comparing the effects on folding rates of these substitutions with database-derived local structure propensity values, we have determined that this position adopts a non-native backbone conformation in the folding transition state. This result is surprising because this position displays a high and canonical Phi-value of 0.7. This work emphasizes the potential role of non-native conformations in folding pathways, and demonstrates that even positions displaying high and canonical Phi- values may, nevertheless, adopt a non-native conformation in the transition state. Proteins 2012. (c) 2012 Wiley-Liss, Inc.

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Comparison of end-point continuum-solvation methods for the calculation of protein-ligand binding free energies.

Publication Date: 2012 Jan 9 PMID: 22274991
Authors: Genheden, S. - Ryde, U.
Journal: Proteins

We have compared the predictions of ligand-binding affinities from several methods based on end-point molecular dynamics simulations and continuum solvation, i.e. methods related to MM/PBSA (molecular mechanics combined with Poisson-Boltzmann and surface area solvation). Two continuum-solvation models were considered, viz. the Poisson-Boltzmann (PB) and generalised Born (GB) approaches. The non-electrostatic energies were also obtained in two different ways, viz. either from the sum of the bonded, van der Waals, non-polar solvation energies, and entropy terms (as in MM/PBSA), or from the scaled protein-ligand van der Waals interaction energy (as in the linear interaction energy approach, LIE). Three different approaches to calculate electrostatic energies were tested, viz. the sum of electrostatic interaction energies and polar solvation energies, obtained either from a single simulation of the complex or from three independent simulations of the complex, the free protein, and the free ligand, or the linear-response approximation (LRA). Moreover, we investigated the effect of scaling the electrostatic interactions by an effective internal dielectric constant of the protein (epsilon(int) ). All these methods were tested on the binding of seven biotin analogues to avidin and nine 3-amidinobenzyl-1H-indole-2-carboxamide inhibitors to factor Xa. For avidin, the best results were obtained with a combination of the LIE non-electrostatic energies with the MM+GB electrostatic energies from a single simulation, using epsilon(int) = 4. For fXa, standard MM/GBSA, based on one simulation and using epsilon(int) = 4-10 gave the best result. The optimum internal dielectric constant seems to be slightly higher with PB than with GB solvation. Proteins 2012. (c) 2012 Wiley-Liss, Inc.

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The chaperone activity of alpha-synuclein: Utilizing deletion mutants to map its interaction with target proteins.

Publication Date: 2012 Jan 11 PMID: 22274962
Authors: Rekas, A. - Ahn, K. J. - Kim, J. - Carver, J. A.
Journal: Proteins

alpha-Synuclein is the principal component of the Lewy body deposits that are characteristic of Parkinson's disease. In vivo, and under physiological conditions in vitro, alpha-synuclein aggregates to form amyloid fibrils, a process that is likely to be associated with the development of Parkinson's disease. alpha-Synuclein also possesses chaperone activity to prevent the precipitation of amorphously aggregating target proteins, as demonstrated in vitro. alpha-Synuclein is an intrinsically disordered (i.e. unstructured) protein of 140 amino acids in length, and therefore studies on its fragments can be correlated directly to the functional role of these regions in the intact protein. In this study, the fragment containing residues 61-140 (alpha-syn(61-140)) was observed to be highly amyloidogenic and was as effective a chaperone in vitro as the full-length protein, while the N- and C-terminal fragments alpha-syn(1-60) and alpha-syn(96-140) had no intrinsic chaperone activity. Interestingly, full-length fibrillar alpha-synuclein had greater chaperone-activity than non-fibrillar alpha-synuclein. It is concluded that the amyloidogenic NAC region (residues 61-95) contains the chaperone-binding site which is optimized for target protein binding as a result of its beta-sheet formation and/or ordered aggregation by alpha-synuclein. On the other hand, the first 60 residues of alpha-synuclein modulate the protein's chaperone-active site, while at the same time protecting alpha-synuclein from fibrillation. On its own, however, this fragment (alpha-syn(1-60)) had a tendency to aggregate amorphously. As a result of this study, the functional roles of the various regions of alpha-synuclein in its chaperone activity have been delineated. Proteins 2012. (c) 2012 Wiley-Liss, Inc.

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Engineering proteins with enhanced mechanical stability by force specific sequence motifs.

Publication Date: 2012 Jan 9 PMID: 22274941
Authors: Lu, W. - Negi, S. - Oberhauser, A. F. - Braun, W.
Journal: Proteins

Use of atomic force microscopy (AFM) has recently led to a better understanding of the molecular mechanisms of the unfolding process by mechanical forces; however, the rational design of novel proteins with specific mechanical strength remains challenging. We have approached this problem from a new perspective that generates linear physical-chemical properties (PCP) motifs from a limited AFM data set. Guided by our linear sequence analysis we designed and analyzed four new mutants of the titin I1 domain with the goal of increasing the domain's mechanical strength. All four mutants could be cloned and expressed as soluble proteins. AFM data indicate that at least two of the mutants have increased molecular mechanical strength. This observation suggests that the PCP method is useful to graft sequences specific for high mechanical stability to weak proteins to increase their mechanical stability, and represents an additional tool in the design of novel proteins besides steered molecular dynamics calculations, coarse grained simulations and phi-value analysis of the transition state. Proteins 2012. (c) 2012 Wiley-Liss, Inc.

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Large-scale conformational change in the mammalian prion protein massive conformation change in the prion protein.

Publication Date: 2012 Jan 9 PMID: 22274922
Authors: Singh, J. P. - Whitford, P. C. - Hayre, N. R. - Onuchic, J. - Cox, D. L.
Journal: Proteins

We employ all-atom structure-based models with a forcefield with multiple energetic basins for the C-terminal (residues 166-226) of the mammalian prion protein. One basin represents the known alpha-helical (alphaH) structure while the other represents the same residues in a left-handed beta helical (LHBH) conformation. The LHBH structure has been proposed to help describe one class of in vitro grown fibrils, as well as possibly self-templating the conversion of normal cellular prion protein to the infectious form. Yet, it is unclear how the protein may make this global rearrangement. Our results demonstrate that the conformation changes are not strongly limited by large-scale geometry modification and that there may exist an overall preference for the LHBH conformation. Furthermore, our model presents novel intermediate trapping conformations with twisted LHBH structure. Proteins 2012. (c) 2012 Wiley-Liss, Inc.

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