Annotation and Curation of the Protein Data Bank

The Protein Data Bank (PDB) is the worldwide repository for experimentally determined 3D structures of biological macromolecules. Established in 1971 with just seven structures, it presently includes more than 56,000 entries. To maintain the highest standards in curation and processing, the members of the worldwide Protein Data Bank (wwPDB) collaborate in data annotation and the development of procedures, tools, and resources. Annotation-related issues, particularly those impacted by new developments
in structural biology, are critically reviewed at in-person and virtual meetings regularly and frequently. Comprehensive documentation of the procedures, formats, and related data dictionaries used in data annotation are available at the wwPDB website(www.wwpdb.org).

Mindful of the impact that changes in annotation procedures or data format may have on users, changes are carefully managed and communicated in a timely fashion. In cases involving complex scientific or policy issues, input is sought from advisory committees, standing task forces, experimental method developers, and community experts. This is exemplified by creation of the recently-released version of the PDB archive which updates and further standardizes database references, small molecule chemistry, biological assemblies, and active sites

Combining intracellular selection with protein-fragment complementation to derive A interacting peptides

Aggregation of the β-amyloid (Aβ) peptide into toxic oligomers is considered the primary event in the pathogenesis of Alzheimer's disease. Previously generated peptides and mimetics designed to bind to amyloid fibrils have encountered problems in solubility, protease susceptibility and the population of small soluble toxic oligomers oligomers. We present a new method that opens the possibility of deriving new amyloid inhibitors. The intracellular protein-fragment complementation assay (PCA) approach uses a semi-rational design approach to generate peptides capable of binding to Aβ. Peptide libraries are based on Aβ regions responsible for instigating amyloidosis, with screening and selection occurring entirely inside Escherichia coli. Successfully selected peptides must therefore bind Aβ and recombine an essential enzyme while permitting bacterial cell survival. No assumptions are made regarding the mechanism of action for selected binders. Biophysical characterisation demonstrates that binding induces a noticeable reduction in amyloid. Therefore, this amyloid-PCA approach may offer a new pathway for the design of effective inhibitors against the formation of amyloid in general

Minimum energy configurations of the 2-dimensional HP-model of proteins by self-organizing networks

We use self-organizing maps (SOM) as an efficient tool to find the minimum energy configurations of the 2-dimensional HP-models of proteins. The usage of the SOM for the protein folding problem is similar to that for the Traveling Salesman Problem. The lattice nodes represent the cities whereas the neurons in the network represent the amino acids moving towards the closest cities, subject to the HH interactions. The valid path that maximizes the HH contacts corresponds to the minimum energy configuration of the protein. We report promising results for the cases when the protein completely fills a lattice and discuss the current problems and possible extensions. In all the test sequences up to 36 amino acids, the algorithm was able to find the global minimum and its degeneracies

Directed evolution converts subtilisin E into a functional equivalent of thermitase

We used directed evolution to convert Bacillus subtilis subtilisin E into an enzyme functionally equivalent to its thermophilic homolog thermitase from Thermoactinomyces vulgaris. Five generations of random mutagenesis, recombination and screening created subtilisin E 5-3H5, whose half-life at 83°C (3.5 min) and temperature optimum for activity (Topt, 76°C) are identical with those of thermitase. The Topt of the evolved enzyme is 17°C higher and its half-life at 65°C is >200 times that of wild-type subtilisin E. In addition, 5-3H5 is more active towards the hydrolysis of succinyl-Ala-Ala-Pro-Phe-p-nitroanilide than wild-type at all temperatures from 10 to 90°C. Thermitase differs from subtilisin E at 157 amino acid positions. However, only eight amino acid substitutions were sufficient to convert subtilisin E into an enzyme equally thermostable. The eight substitutions, which include known stabilizing mutations (N218S, N76D) and also several not previously reported, are distributed over the surface of the enzyme. Only two (N218S, N181D) are found in thermitase. Directed evolution provides a powerful tool to unveil mechanisms of thermal adaptation and is an effective and efficient approach to increasing thermostability without compromising enzyme activity

Epitope mapping using mRNA display and a unidirectional nested deletion library

In vitro selection targeting an anti-polyhistidine monoclonal antibody was performed using mRNA display with a random, unconstrained 27-mer peptide library. After six rounds of selection, epitope-like peptides were identified that contain two to five consecutive, internal histidines and are biased for arginine residues, without any other identifiable consensus. The epitope was further refined by constructing a high-complexity, unidirectional fragment library from the final selection pool. Selection by mRNA display minimized the dominant peptide from the original selection to a 15-residue functional sequence (peptide Cmin: RHDAGDHHHHHGVRQ; K-D = 38 nM). Other peptides recovered from the fragment library selection revealed a separate consensus motif (ARRXA) C-terminal to the histidine track. Kinetics measurements made by surface plasmon resonance, using purified Fab (antigen-binding fragment) to prevent avidity effects, demonstrate that the selected peptides bind with 10- to 75-fold higher affinities than a hexahistidine peptide. The highest affinity peptides (K-D approximate to 10 nM) encode both a short histidine track and the ARRXA motif, suggesting that the motif and other flanking residues make important contacts adjacent to the core polyhistidine-binding site and can contribute > 2.5 kcal/mol of binding free energy. The fragment library construction methodology described here is applicable to the development of high-complexity protein or cDNA expression libraries for the identification of protein-protein interaction domains

Homology modelling of transferrin-binding protein A from Neisseria meningitidis

Neisseria meningitidis, a causative agent of bacterial meningitis, obtains transferrin-bound iron by expressing two outer membrane located transferrin-binding proteins, TbpA and TbpB. TbpA is thought to be an integral outer membrane pore that facilitates iron uptake. Evidence suggests that TbpA is a useful antigen for inclusion in a vaccine effective against meningococcal disease, hence the identification of regions involved in ligand binding is of paramount importance to design strategies to block uptake of iron. The protein shares sequence and functional similarities to the Escherichia coli siderophore receptors FepA and FhuA, whose structures have been determined. These receptors are composed of two domains, a 22-stranded b-barrel and an N-terminal plug region that sits within the barrel and occludes the transmembrane pore. A three-dimensional TbpA model was constructed using FepA and FhuA structural templates, hydrophobicity analysis and homology modelling. TbpA was found to possess a similar architecture to the siderophore receptors. In addition to providing insights into the highly immunogenic nature of TbpA and allowing the prediction of potentially important ligandbinding epitopes, the model also reveals a narrow channel through its entire length. The relevance of this channel and the spatial arrangement of external loops, to the mechanism of iron translocation employed by TbpA is discussed

Influenza Evolution and H3N2 Vaccine Effectiveness, with Application to the 2014/2015 Season

Influenza A is a serious disease that causes significant morbidity and mortality, and vaccines against the seasonal influenza disease are of variable effectiveness. In this paper, we discuss use of the $p_{\rm epitope}$ method to predict the dominant influenza strain and the expected vaccine effectiveness in the coming flu season. We illustrate how the effectiveness of the 2014/2015 A/Texas/50/2012 [clade 3C.1] vaccine against the A/California/02/2014 [clade 3C.3a] strain that emerged in the population can be estimated via pepitope. In addition, we show by a multidimensional scaling analysis of data collected through 2014, the emergence of a new A/New Mexico/11/2014-like cluster [clade 3C.2a] that is immunologically distinct from the A/California/02/2014-like strains.Comment: 19 pages, 4 figure

Detecting similarities among distant homologous proteins by comparison of domain flexibilities

Aim of this work is to assess the informativeness of protein dynamics in the detection of similarities among distant homologous proteins. To this end, an approach to perform large-scale comparisons of protein domain flexibilities is proposed. CONCOORD is confirmed as a reliable method for fast conformational sampling. The root mean square fluctuation of alpha carbon positions in the essential dynamics subspace is employed as a measure of local flexibility and a synthetic index of similarity is presented. The dynamics of a large collection of protein domains from ASTRAL/SCOP40 is analyzed and the possibility to identify relationships, at both the family and the superfamily levels, on the basis of the dynamical features is discussed. The obtained picture is in agreement with the SCOP classification, and furthermore suggests the presence of a distinguishable familiar trend in the flexibility profiles. The results support the complementarity of the dynamical and the structural information, suggesting that information from dynamics analysis can arise from functional similarities, often partially hidden by a static comparison. On the basis of this first test, flexibility annotation can be expected to help in automatically detecting functional similarities otherwise unrecoverable. © 2007 The Author(s)

Computationally designed variants of Escherichia coli chorismate mutase show altered catalytic activity

Computational protein design methods were used to predict five variants of monofunctional Escherichia coli chorismate mutase expected to maintain catalytic activity. The variants were tested experimentally and three active site mutants exhibited catalytic activity similar to or greater than the wild-type enzyme. One mutant, Ala32Ser, showed increased catalytic efficiency

Influence of mutations of Val226 on the catalytic rate of haloalkane dehalogenase

Haloalkane dehalogenase converts haloalkanes to their corresponding alcohols. The 3D structure, reaction mechanism and kinetic mechanism have been studied. The steady state kcat with 1,2-dichloroethane and 1,2-dibromoethane is limited mainly by the rate of release of the halide ion from the buried active-site cavity. During catalysis, the halogen that is cleaved off (Clα) from 1,2-dichloroethane interacts with Trp125 and the Clβ interacts with Phe172. Both these residues have van der Waals contacts with Val226. To establish the effect of these interactions on catalysis, and in an attempt to change enzyme activity without directly mutating residues involved in catalysis, we mutated Val226 to Gly, Ala and Leu. The Val226Ala and Val226Leu mutants had a 2.5-fold higher catalytic rate for 1,2-dibromoethane than the wild-type enzyme. A pre-steady state kinetic analysis of the Val226Ala mutant enzyme showed that the increase in kcat could be attributed to an increase in the rate of a conformational change that precedes halide release, causing a faster overall rate of halide dissociation. The kcat for 1,2-dichloroethane conversion was not elevated, although the rate of chloride release was also faster than in the wild-type enzyme. This was caused by a 3-fold decrease in the rate of formation of the alkyl-enzyme intermediate for 1,2-dichloroethane. Val226 seems to contribute to leaving group (Clα or Brα) stabilization via Trp125, and can influence halide release and substrate binding via an interaction with Phe172. These studies indicate that wild-type haloalkane dehalogenase is optimized for 1,2-dichloroethane, although 1,2-dibromoethane is a better substrate.