Proteins Structure Function and Bioinformatics (Protein Struct Funct Genet)

Publications in this journal

• Article: Substrate versus inhibitor dynamics of P-glycoprotein.

  Jerome Ma, Philip C Biggin
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  ABSTRACT: By far the most studied multidrug resistance protein is P-glycoprotein. Despite recent structural data, key questions about its function remain. P-glycoprotein (P-gp) is flexible and undergoes large conformational changes as part of its function and in this respect details not only of the export cycle but also the recognition stage are currently lacking. Given the flexibility, molecular dynamics (MD) simulations provide an ideal tool to examine this aspect in detail. We have performed MD simulations to examine the behaviour of P-gp. In agreement with previous reports, we found that P-gp undergoes large conformational changes which tended to result in the nucleotide-binding domains coming closer together. In all simulations, the approach of the NBDs was asymmetrical in agreement with previous observations for other ABC transporter proteins. To validate the simulations, we make extensive comparison to the available cross-linking data. Our results show very good agreement with the available data. We then went on to compare the influence of inhibitor compounds with simulations of a substrate (daunorubicin) bound. Our results suggest that inhibitors may work by keeping the NBDs apart and thus preventing ATP-hydrolysis. On the other hand, repeat simulations of daunorubicin (substrate) in one particular binding pose suggest that the approach of the NBDs is not impaired and that the structure would be still be competent to perform ATP hydrolysis, thus providing a model for inhibition or substrate transport. Finally we compare the latter to earlier QSAR data to provide a model for the first part of substrate transport within P-gp. © Proteins 2013;. © 2013 Wiley Periodicals, Inc.
  Proteins Structure Function and Bioinformatics 05/2013;
• Article: Structure based engineering of streptavidin monomer with a reduced biotin dissociation rate.

  Daniel Demonte, Eric J Drake, Kok Hong Lim, Andrew M Gulick, Sheldon Park
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  ABSTRACT: We recently reported the engineering of monomeric streptavidin, mSA, corresponding to a single subunit of wild type (wt) streptavidin tetramer. The monomer was designed by homology modeling, in which the streptavidin and rhizavidin sequences were combined to engineer a high affinity binding pocket containing residues from a single subunit only. Although mSA is stable and binds biotin with nanomolar affinity, its fast off rate (koff ) creates practical challenges during applications. We obtained a 1.9 Å crystal structure of mSA bound to biotin to understand their interaction in detail, and used the structure to introduce targeted mutations to improve its binding kinetics. F43 of shwanavidin forms a hydrophobic lid that is important for biotin binding. However, the T48F mutation in mSA, which introduces a comparable hydrophobic lid only results in a modest 20 - 40% improvement in the measured koff . On the other hand, introducing the S25H mutation near the bicyclic ring of bound biotin increases the dissociation half life (t½ ) from 11 min to 83 min at 20 °C. Molecular dynamics (MD) simulations suggest that H25 stabilizes the binding loop L3,4 by interacting with A47, and protects key intermolecular hydrogen bonds by limiting solvent entry into the binding pocket. Concurrent T48F or T48W mutation clashes with H25 and partially abrogates the beneficial effects of H25. Taken together, this study suggests that stabilization of the binding loop and solvation of the binding pocket are important determinants of the dissociation kinetics in mSA. © Proteins 2013;. © 2013 Wiley Periodicals, Inc.
  Proteins Structure Function and Bioinformatics 05/2013;
• Article: Ab initio folding of extended α-helix: A theoretical study about the role of electrostatic polarization in the folding of helical structures.

  Raudah Lazim, Caiyi Wei, Tiedong Sun, Dawei Zhang
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  ABSTRACT: In this work, we report the ab initio folding of three different extended helical peptides namely 2khk, N36 and C34 through conventional molecular dynamics simulation at room temperature using implicit solvation model. Employing adaptive hydrogen bond specific charge (AHBC) scheme to account for the polarization effect of hydrogen bonds established during the simulation, the effective folding of the three extended helices were observed with best backbone RMSDs in comparison to the experimental structures over the helical region determined to be 1.30 Å for 2khk, 0.73 Å for N36 and 0.72 Å for C34. In this study, 2khk will be used as a benchmark case serving as a means to compare the ability of polarized (AHBC) and non-polarized force field in the folding of an extended helix. Analyses conducted revealed the ability of the AHBC scheme in effectively folding the extended helix by promoting helix growth through the stabilization of backbone hydrogen bonds upon formation during the folding process. Similar observations were also noted when AHBC scheme was employed during the folding of C34 and N36. However, under Amber03 force field, helical structures formed during the folding of 2khk was not accompanied by stabilization thus highlighting the importance of electrostatic polarization in the folding of helical structures. © Proteins 2013;. © 2013 Wiley Periodicals, Inc.
  Proteins Structure Function and Bioinformatics 05/2013;
• Article: Crystal structure of the cataract-causing P23T γD-crystallin mutant.

  Fangling Ji, Leonardus M I Koharudin, Jinwon Jung, Angela M Gronenborn
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  ABSTRACT: Up to now, efforts to crystallize the cataract-associated P23T mutant of human γD-crystallin have not been successful. Therefore, insights into the light scattering mechanism of this mutant have been exclusively obtained from solution work. Here we present the first crystal structure of the P23T mutant at 2.5Å resolution. The protein exhibits essentially the same overall structure as seen for the wild-type protein. Based on our structural data, we confirm that no major conformational changes are caused by the mutation, and that solution phase properties of the mutant appear exclusively associated with cataract formation. © Proteins 2013;. © 2013 Wiley Periodicals, Inc.
  Proteins Structure Function and Bioinformatics 05/2013;
• Article: Alternative zinc-binding sites explain the redox sensitivity of zinc-containing anti-sigma factors.

  Lim Heo, Yoo-Bok Cho, Myeong Sup Lee, Jung-Hye Roe, Chaok Seok
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  ABSTRACT: Certain bacterial zinc-containing anti-sigma (ZAS) factors respond sensitively to thiol-induced oxidative stress by undergoing conformational changes, which in turn reduce binding affinities for their cognate sigma factors. This redox sensitivity provides a mechanism for coping with oxidative stress by activating the transcription of antioxidant genes. Not all ZAS proteins are redox-sensitive, but the mechanism of redox sensitivity is not fully understood. Here we propose that alternative zinc-binding sites determine redox sensitivity. To support this proposal, we performed protein modeling and zinc docking on redox-sensitive and redox-insensitive ZAS proteins complexed with their cognate sigma factors. At least one strong alternative zinc-binding pocket was detected for all known redox-sensitive ZAS factors in actinomycetes, while no strong alternative zinc-binding pocket was identified in redox-insensitive ZAS factors, except for one controversial case. This hypothesis of alternative zinc-binding sites can also explain residue-specific contributions to the redox sensitivity of RsrA, a redox-sensing ZAS protein from Streptomyces coelicolor, for which alanine mutagenesis experiments are available. Our results suggest a mechanistic model for redox sensitivity as follows: zinc ion can probabilistically occupy multiple sites in redox-sensitive ZAS proteins, increasing the susceptibility of zinc-coordinating cysteine residues to oxidation. This picture of probabilistic zinc occupation agrees with a previous structure and energy analysis on zinc finger proteins, and thus it may be more widely applicable to other classes of reactive zinc-binding proteins. © Proteins 2013;. © 2013 Wiley Periodicals, Inc.
  Proteins Structure Function and Bioinformatics 05/2013;
• Article: Molecular function prediction for a family exhibiting evolutionary tendencies towards substrate specificity swapping: Recurrence of tyrosine aminotransferase activity in the Iα subfamily.

  Kathryn E Muratore, Barbara E Engelhardt, John R Srouji, Michael I Jordan, Steven E Brenner, Jack F Kirsch
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  ABSTRACT: The subfamily Iα aminotransferases are typically categorized as having narrow specificity towards carboxylic amino acids (AATases), or broad specificity that includes aromatic amino acid substrates (TATases). Because of their general role in central metabolism and, more specifically, their association with liver-related diseases in humans, this subfamily is biologically interesting. The substrate specificities for only a few members of this subfamily have been reported, and the reliable prediction of substrate specificity from protein sequence has remained elusive. In this study, a diverse set of aminotransferases was chosen for characterization based on a scoring system that measures the sequence divergence of the active site. The enzymes that were experimentally characterized include both narrow-specificity AATases and broad-specificity TATases, as well as AATases with broader-specificity and TATases with narrower-specificity than the previously known family members. Molecular function and phylogenetic analyses underscored the complexity of this family's evolution as the TATase function does not follow a single evolutionary thread, but rather appears independently multiple times during the evolution of the subfamily. The additional functional characterizations described in this paper, alongside a detailed sequence and phylogenetic analysis, provide some novel clues to understanding the evolutionary mechanisms at work in this family. © Proteins 2013;. © 2013 Wiley Periodicals, Inc.
  Proteins Structure Function and Bioinformatics 05/2013;
• Article: In vitro and in vivo characterization of designed immunogens derived from the CD-helix of the stem of influenza hemagglutinin.

  V Vamsee Aditya Mallajosyula, Michael Citron, Xianghan Lu, Jan Ter Meulen, Raghavan Varadarajan, Xiaoping Liang
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  ABSTRACT: The conserved 'stem' domain of influenza virus hemagglutinin (HA) is a target for broadly neutralizing antibodies (bnAbs) and a potential vaccine antigen for induction of hetero-subtypic protection. The epitope of 12D1, a previously reported bnAb neutralizing several H3 subtype influenza strains, was putatively mapped to residues 76-106 of the CD-helix, also referred to as long alpha helix (LAH) of the HA stem. A peptide derivative consisting of wt-LAH residues 76-130 conjugated to keyhole limpet hemocyanin was previously shown to confer robust protection in mice against challenge with influenza strains of subtypes H3, H1 and H5 which motivated the present study. We report the design of multiple peptide derivatives of LAH with or without heterologous trimerization sequences and show that several of these are better folded than wt-LAH. However, in contrast to the previous study immunization of mice with wt-LAH resulted in negligible protection against a lethal homologous virus challenge, while some of the newly designed immunogens could confer weak protection. Combined with structural analysis of HA, our data suggest that in addition to LAH, other regions of HA are likely to significantly contribute to the epitope for 12D1 and will be required to elicit robust protection. In addition, a dynamic, flexible conformation of isolated LAH peptide may be required for eliciting a functional anti-viral response. © Proteins 2013;. © 2013 Wiley Periodicals, Inc.
  Proteins Structure Function and Bioinformatics 04/2013;
• Article: Crystal structure of deltarhodopsin-3 from haloterrigena thermotolerans.

  Jin Zhang, Katsuhide Mizuno, Yuki Murata, Hideaki Koide, Midori Murakami, Kunio Ihara, Tsutomu Kouyama
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  ABSTRACT: Deltarhodopsin, a new member of the microbial rhodopsin family, functions as a light-driven proton pump. Here, we report the three-dimensional structure of deltarhodopsin (dR3) from Haloterrigena thermotolerans at 2.7 Å resolution. A crystal belonging to space group R32 (a, b = 111.71 Å, c = 198.25 Å) was obtained by the membrane fusion method. In this crystal, dR3 forms a trimeric structure as observed for bacteriorhodopsin (bR). Structural comparison of dR with bR showed that the inner part (the proton release and uptake pathways) is highly conserved. Meanwhile, residues in the protein-protein contact region are largely altered so that the diameter of the trimeric structure at the cytoplasmic side is noticeably larger in dR3. Unlike bR, dR3 possesses a helical segment at the C-terminal region that fills the space between the AB and EF loops. A significant difference is also seen in the FG loop, which is one residue longer in dR3. Another peculiar property of dR3 is a highly crowded distribution of positively charged residues on the cytoplasmic surface, which may be relevant to a specific interaction with some cytoplasmic component. © Proteins 2013;. © 2013 Wiley Periodicals, Inc.
  Proteins Structure Function and Bioinformatics 04/2013;
• Article: Crystal structure of the protein from Arabidopsis thaliana gene At5g06450, a putative DnaQ-like exonuclease domain-containing protein with homohexameric assembly.

  David W Smith, Mi Ra Han, Joon Sung Park, Kyung Rok Kim, Taeho Yeom, Ji Yeon Lee, Do Jin Kim, Craig A Bingman, Hyun-Jung Kim, Kyubong Jo, Byung Woo Han, George N Phillips
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  ABSTRACT: Arabidopsis thaliana gene At5g06450 encodes a putative DnaQ-like 3'-5' exonuclease domain-containing protein (AtDECP). The DnaQ-like 3'-5' exonuclease domain is often found as a proofreading domain of DNA polymerases. The overall structure of AtDECP adopts an RNase H fold that consists of a mixed β-sheet flanked by α-helices. Interestingly, AtDECP forms a homohexameric assembly with a central 6-fold symmetry, generating a central cavity. The ring-shaped structure and comparison with WRN-exo, the best structural homologue of AtDECP, suggest a possible mechanism for implementing its exonuclease activity using positively charged patch on the N-terminal side of the homohexameric assembly. The homohexameric structure of AtDECP provides unique information about the interaction between the DnaQ-like 3'-5' exonuclease and its substrate nucleic acids. © Proteins 2013;. © 2013 Wiley Periodicals, Inc.
  Proteins Structure Function and Bioinformatics 04/2013;
• Article: Probing a continuum of macro-molecular assembly models with graph templates of complexes.

  Tom Dreyfus, Valérie Doye, Frédéric Cazals
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  ABSTRACT: Reconstruction by data integration is an emerging trend to reconstruct large protein assemblies, but uncertainties on the input data yield average models whose quantitative interpretation is challenging. This paper presents methods to probe fuzzy models of large assemblies against atomic resolution models of sub-systems. Consider a Toleranced Model (TOM) of a macro-molecular assembly, namely a continuum of nested shapes representing the assembly at multiple scales. Also consider a template namely an atomic resolution 3D model of a sub-system (a complex) of this assembly. We present graph-based algorithms performing a multi-scale assessment of the complexes of the TOM, by comparing the pairwise contacts which appear in the TOM against those of the template. We apply this machinery on TOM derived from an average model of the Nuclear Pore Complex, to explore the connections among members of its well-characterized Y-complex. The software implementing the algorithms of this paper, called GRAPH_MATCHER, is available from the VORATOM suite, see http://team.inria.fr/abs/software/voratom © Proteins 2013;. © 2013 Wiley Periodicals, Inc.
  Proteins Structure Function and Bioinformatics 04/2013;
• Article: Ranking multiple docking solutions based on the conservation of inter-residue contacts.

  Romina Oliva, Anna Vangone, Luigi Cavallo
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  ABSTRACT: Molecular docking is the method of choice for investigating the molecular basis of recognition in a large number of functional protein complexes. However, correctly scoring the obtained docking solutions (decoys) to rank native-like conformations in the top positions is still an open problem. Herein we present CONS-RANK, a simple and effective tool to rank multiple docking solutions, which relies on the conservation of inter-residue contacts in the analysed decoys ensemble. First it calculates a conservation rate for each inter-residue contact, then it ranks decoys according to their ability to match the more frequently observed contacts. We applied CONS-RANK to 102 targets from three different benchmarks, RosettaDock, DOCKGROUND and CAPRI. The method performs consistently well, both in terms of native-like solutions ranked in the top positions and of values of the area under the receiver operating characteristic (ROC) curve. Its ideal application is to solutions coming from different docking programs and procedures, as in the case of CAPRI targets. For all the analysed CAPRI targets where a comparison is feasible, CONS-RANK outperforms the CAPRI scorers. The fraction of native-like solutions in the top ten positions in the RosettaDock, DOCKGROUND and CAPRI benchmarks is enriched on average by a factor of 3.0, 1.9 and 9.9, respectively. Interestingly, CONS-RANK is also able to specifically single out the high/medium quality solutions from the docking decoys ensemble: it ranks 46.2% and 70.8% of the total high/medium quality solutions available for the RosettaDock and CAPRI targets, respectively, within the top twenty positions. © Proteins 2013;. © 2013 Wiley Periodicals, Inc.
  Proteins Structure Function and Bioinformatics 04/2013;
• Article: Crystal structure of human cytosolic aspartyl-tRNA synthetase, a component of multi-tRNA synthetase complex.

  Kyung Rok Kim, Sang Ho Park, Hyoun Sook Kim, Kyung Hee Rhee, Byung-Gyu Kim, Dae Gyu Kim, Mi Seul Park, Hyun-Jung Kim, Sunghoon Kim, Byung Woo Han
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  ABSTRACT: Human cytosolic aspartyl-tRNA synthetase (DRS) catalyzes the attachment of the amino acid aspartic acid to its cognate tRNA and it is a component of the multi-tRNA synthetase complex (MSC) which has been known to be involved in unexpected signaling pathways. Here, we report the crystal structure of DRS at 2.25 Å resolution. DRS is a homodimer with a dimer interface 3,750.5 Å(2) which comprises of 16.6% of the monomeric surface area. Our structure reveals the C-terminal end of the N-helix which is considered as a unique addition in DRS, and its conformation further supports the switching model of the N-helix for the transfer of tRNA(Asp) to elongation factor 1α. From our analyses of the crystal structure and post-translational modification of DRS, we suggest that the phosphorylation of Ser146 provokes the separation of DRS from the MSC and provides the binding site for an interaction partner with unforeseen functions. © Proteins 2013;. © 2013 Wiley Periodicals, Inc.
  Proteins Structure Function and Bioinformatics 04/2013;
• Article: Capturing protein sequence-structure specificity using computational sequence design.

  Paul Mach, Patrice Koehl
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  ABSTRACT: It is well known that protein fold recognition can be greatly improved if models for the underlying evolution history of the folds are taken into account. The improvement however only exists if such evolutionary information is available. To circumvent this limitation for protein families that only have a small number of representatives in current sequence databases, we follow an alternate approach in which the benefits of including evolutionary information can be recreated by using sequences generated by computational protein design algorithms. We explore this strategy on a large database of protein templates with 1747 members from different protein families. An automated method is used to design sequences for these templates. We use the backbones from the experimental structures as fixed templates, thread sequences on these backbones using a self consistent mean field approach, and score the fitness of the corresponding model using a semi-empirical physical potential. Sequences designed for one template are translated into a HMM-based profile. We describe the implementation of this method, the optimization of its parameters, and its performance. When the native sequences of the protein templates were tested against the library of these profiles, the class, fold, and family memberships of a large majority (over 90%) of these sequences were correctly recognized for an E-value threshold of 1. In contrast, when homologous sequences were tested against the same library, a much smaller fraction (35%) of sequences were recognized; The SCOP families corresponding to these sequences however are correctly recognized (with an accuracy > 88%). © Proteins 2013;. © 2013 Wiley Periodicals, Inc.
  Proteins Structure Function and Bioinformatics 04/2013;
• Article: Analyzing the effect of homogeneous frustration in protein folding.

  V G Contessoto, D T Lima, R J Oliveira, A T Bruni, J Chahine, V B P Leite
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  ABSTRACT: The energy landscape theory has been an invaluable theoretical framework in the understanding of biological processes such as protein folding, oligomerization and functional transitions. According to the theory, the energy landscape of protein folding is funneled towards the native state, a conformational state that is consistent with the principle of minimal frustration. It has been accepted that real proteins are selected through natural evolution satisfying the minimum frustration criterion. However, there is evidence that a low degree of frustration accelerates folding. We examined the interplay between topological and energetic protein frustration. We employed a Cα structure-based model for simulations with a controlled nonspecific energetic frustration added to the potential energy function. Thermodynamics and kinetics of a group of nineteen proteins are completely characterized as a function of increasing level of energetic frustration. We observed two well separated groups of proteins: one group where a little frustration enhances folding rates to an optimal value and another where any energetic frustration slows down folding. Protein energetic frustration regimes and their mechanisms are explained by the role of nonnative contact interactions in different folding scenarios. These findings strongly correlate with the protein free energy folding barrier and the absolute contact order parameters. These computational results are corroborated by Principal Component Analysis (PCA) and Partial Least Square (PLS) techniques. One simple theoretical model is proposed as a useful tool for experimentalists to predict the limits of improvements in real proteins. © Proteins 2013;. © 2013 Wiley Periodicals, Inc.
  Proteins Structure Function and Bioinformatics 04/2013;
• Article: Heterogeneity of single molecule FRET signals reveals multiple active ribosome subpopulations.

  Yuhong Wang, Ming Xiao, Yue Li
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  ABSTRACT: Single molecule methods have revealed that heterogeneity is common in biological systems. However, interpretations of the complex signals are challenging. By tracking the fluorescence resonance energy transfer (FRET) signals between the A-site tRNA and L27 protein in single ribosomes, we attempt to develop a qualitative method to subtract the inherent patterns of the heterogeneous single molecule FRET data. Seven ribosome subpopulations are identified using this method and spontaneous exchanges among these subpopulations are observed. All of the pre-translocation subpopulations are competent in real-time translocation, but via distinguished pathways. These observations suggest that the ribosome may function through multiple reaction pathways. © Proteins 2013;. © 2013 Wiley Periodicals, Inc.
  Proteins Structure Function and Bioinformatics 04/2013;
• Article: Impact of the K24N mutation on the transactivation domain of p53 and its binding to MDM2.

  Yingqian Ada Zhan, Hongwei Wu, Anne T Powell, Gary W Daughdrill, F Marty Ytreberg
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  ABSTRACT: The level of the p53 transcription factor is negatively regulated by the E3 ubiguitin ligase murine double minute clone 2 (MDM2). The interaction between p53 and MDM2 is essential for the maintenance of genomic integrity for most eukaryotes. Previous structural studies revealed that MDM2 binds to p53 transactivation domain (p53TAD) from residues 17 to 29. The K24N mutation of p53TAD changes a lysine at position 24 to an asparagine. This mutation occurs naturally in the bovine family and is also found in a rare form of human gestational cancer called choriocarcinoma. In this study we have investigated how the K24N mutation affects the affinity, structure, and dynamics of p53TAD binding to MDM2. Nuclear magnetic resonance studies of p53TAD show the K24N mutant is more flexible and has less transient helical secondary structure than the wildtype. Isothermal titration calorimetry measurements demonstrate that these changes in structure and dynamics do not significantly change the binding affinity for p53TAD-MDM2. Finally, free energy perturbation and standard molecular dynamics simulations suggest the negligible affinity change is due to a compensating interaction energy between the K24N mutant and MDM2 when it is bound. Overall, the data suggests that the K24N-MDM2 complex is able to at least partly compensate for an increase in the conformational entropy in unbound K24N with an increase in the bound state electrostatic interaction energy. © Proteins 2013;. © 2013 Wiley Periodicals, Inc.
  Proteins Structure Function and Bioinformatics 04/2013;
• Article: Structural studies of the tethered N-terminus of the Alzheimer's disease Aβ peptide.

  Rebecca M Nisbet, Stewart D Nuttall, Remy Robert, Joanne M Caine, Olan Dolezal, Meghan Hattarki, Lesley A Pearce, Natalia Davydova, Colin L Masters, Jose N Varghese, Victor A Streltsov
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  ABSTRACT: Alzheimer's disease is the most common form of dementia in humans and is related to the accumulation of the amyloid-β (Aβ) peptide and its interaction with metals (Cu, Fe and Zn) in the brain. Crystallographic structural information about Aβ peptide deposits and the details of the metal binding site is limited due to the heterogeneous nature of aggregation states formed by the peptide. Here we present a crystal structure of Aβ residues 1-16 fused to the N-terminus of the E. coli immunity protein Im7, and stabilized with the Fab fragment of the anti-Aβ N-terminal antibody WO2. The structure demonstrates that Aβ residues 10-16, which are not in complex with the antibody, adopt a mixture of local polyproline II (PPII) helix and turn type conformations, enhancing co-operativity between the two adjacent histidine residues His13 and His14. Furthermore, this relatively rigid region of Aβ (residues 10-16) appear as an almost independent unit available for trapping metal ions and provides a rationale for the His13-metal-His14 coordination in the Aβ1-16 fragment implicated in Aβ metal binding. This novel structure therefore has the potential to provide a foundation for investigating the effect of metal ion binding to Aβ and illustrates a potential target for development of future Alzheimer's disease therapeutics aimed at stabilizing the N-terminal monomer structure, in particular residues His13 and His14, and preventing Aβ metal binding-induced neurotoxicity. © Proteins 2013;. © 2013 Wiley Periodicals, Inc.
  Proteins Structure Function and Bioinformatics 04/2013;
• Article: Structure and function of Escherichia coli RimK, an ATP-grasp fold, L-glutamyl ligase enzyme.

  Gengxiang Zhao, Zhongmin Jin, Yanli Wang, Norma M Allewell, Mendel Tuchman, Dashuang Shi
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  ABSTRACT: We report herein the crystal structure of E. coli RimK at 2.85 Å resolution, an enzyme that catalyzes the post-translational addition of up to fifteen C-terminal glutamate residues to ribosomal protein S6. The structure belongs to the ATP-grasp superfamily and is organized as a tetramer, consistent with gel filtration analysis. Each subunit consists of three distinct structural domains and the active site is located in the cleft between these domains. The catalytic reaction appears to occur at the junction between the three domains since ATP binds between the B and C domains, and other substrates bind nearby. © Proteins 2013;. © 2013 Wiley Periodicals, Inc.
  Proteins Structure Function and Bioinformatics 04/2013;
• Article: Structural and functional characterization of solute binding proteins for aromatic compounds derived from lignin: p-coumaric acid and related aromatic acids.

  Kemin Tan, Changsoo Chang, Marianne Cuff, Jerzy Osipiuk, Elizabeth Landorf, Jamey C Mack, Sarah Zerbs, Andrzej Joachimiak, Frank R Collart
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  ABSTRACT: Lignin comprises 15-25% of plant biomass and represents a major environmental carbon source for utilization by soil microorganisms. Access to this energy resource requires the action of fungal and bacterial enzymes to break down the lignin polymer into a complex assortment of aromatic compounds that can be transported into the cells. To improve our understanding of the utilization of lignin by microorganisms, we characterized the molecular properties of solute binding proteins of ATP-binding cassette transporter proteins that interact with these compounds. A combination of functional screens and structural studies characterized the binding specificity of the solute binding proteins for aromatic compounds derived from lignin such as p-coumarate, 3-phenylpropionic acid and compounds with more complex ring substitutions. A ligand screen based on thermal stabilization identified several binding protein clusters that exhibit preferences based on the size or number of aromatic ring substituents. Multiple X-ray crystal structures of protein-ligand complexes for these clusters identified the molecular basis of the binding specificity for the lignin-derived aromatic compounds. The screens and structural data provide new functional assignments for these solute-binding proteins which can be used to infer their transport specificity. This knowledge of the functional roles and molecular binding specificity of these proteins will support the identification of the specific enzymes and regulatory proteins of peripheral pathways that funnel these compounds to central metabolic pathways and will improve the predictive power of sequence-based functional annotation methods for this family of proteins. © Proteins 2013;. © 2013 Wiley Periodicals, Inc.
  Proteins Structure Function and Bioinformatics 04/2013;
• Article: The conserved Arg241-Glu439 salt bridge determines flexibility of the inositol 1,4,5-trisphosphate receptor binding core in the ligand-free state.

  Yoichi Ida, Akinori Kidera
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  ABSTRACT: Inositol 1,4,5-trisphosphate receptor (InsP3 R) is an intracellular Ca(2+) -release channel activated by binding of inositol 1,4,5-trisphosphate (InsP3 ) to the InsP3 binding core (IBC). Structural change in the IBC upon InsP3 binding is the key process in channel pore opening. In this study, we performed molecular dynamics (MD) simulations of the InsP3 -free form of the IBC, starting with removal of InsP3 from the InsP3 -bound crystal structure, and obtained the structural ensemble of the InsP3 -free form of the IBC. The simulation revealed that the two domains of the IBC largely fluctuate around the average structure with the hinge angle opened 17º more than in the InsP3 -bound form, and the twist angle rotated by 45º, forming inter-domain contacts that are different from those in the bound form. The InsP3 binding loop was disordered. The InsP3 -free form thus obtained was reproduced four times in simulations started from a fully extended configuration of the two domains. Simulations beginning with the fully extended form indicated that formation of a salt bridge between Arg241 and Glu439 is crucial for stabilizing the closed form of the two domains. Mutation of Arg241 to Gln prevented formation of the compact structure by the two domains, but the fully flexible domain arrangement was maintained. Thus, the Arg241-Glu439 salt bridge determines the flexibility of the InsP3 -free form of the IBC. © Proteins 2013;. © 2013 Wiley Periodicals, Inc.
  Proteins Structure Function and Bioinformatics 04/2013;