A structural-informatics approach for mining beta-sheets: locating sheets in intermediate-resolution density maps

Here, we report a new computational method, called sheetminer, for mining beta-sheets in the density maps at intermediate resolutions of 6 to 10A. The method employs a multi-step ad hoc morphological analysis of density maps to identify the unique characteristics of beta-sheets. It was tested on density maps from 12 protein crystal structures that were artificially blurred to intermediate resolutions. There are a total of 35 independent beta-sheets with a wide distribution of morphology. The method successfully located 34 of them and missed only one. The method was also applied to an experimental 9A electron cryomicroscopic structure and an 8A X-ray density map. In both cases, the sheet-searching results were found to agree very well with known high-resolution crystal structures. Collectively, these results demonstrate clearly the robustness of sheetminer in locating the regions belonging to beta-sheets in the intermediate-resolution density maps. Furthermore, sheetminer is completely complementary to all other existing computational methods, including helixhunter and threading algorithms. Their combined usage has the potential to significantly enhance the computational modeling capacity for a much more complete interpretation of structural data at intermediate resolutions, from which extraction of functional information would be more effective. This is particularly important in the field of structural genomics, in which the fast screening approach may not always yield crystals that diffract to atomic resolution. An exciting future application of sheetminer is as a valuable tool for revealing the structures of amyloid fibrils that are rich in beta-motifs.     

A Structural-informatics approach for tracing beta-sheets: building pseudo-C(alpha) traces for beta-strands in intermediate-resolution density maps

We report the development of two computational methods to assist density map interpretation at intermediate resolutions: sheettracer for building pseudo-C(alpha) models of beta-sheets, and a deconvolution method for enhancing features attributed to major secondary structural elements. Sheettracer is tightly coupled with sheetminer, which was developed to locate sheet densities in intermediate-resolution density maps. The results from sheetminer are used as inputs to sheettracer, which employs a multi-step ad hoc morphological analysis of sheet densities to trace individual strands of beta-sheets. The methods were tested on simulated density maps from 12 protein crystal structures that represent a reasonably complete sampling of sheet morphology. The sheet-tracing results were quantitatively assessed in terms of sensitivity, specificity and rms deviations. Furthermore, sheettracer and the deconvolution method were rigorously tested on experimental maps of the lambda2 protein of reovirus at resolutions of 7.6A and 11.8A. Our results clearly demonstrate the capability of sheettracer in building pseudo-C(alpha) models of beta-sheets in intermediate-resolution density maps and the power of the deconvolution method in enhancing the performance of sheettracer. These computational methods, along with other related ones, should facilitate recognition and analysis of folding motifs from experimental data at intermediate resolutions.     

Predicted residue–residue contacts can help the scoring of 3D models

During the 7th Critical Assessment of Protein Structure Prediction (CASP7) experiment, it was suggested that the real value of predicted residue–residue contacts might lie in the scoring of 3D model structures. Here, we have carried out a detailed reassessment of the contact predictions made during the recent CASP8 experiment to determine whether predicted contacts might aid in the selection of close‐to‐native structures or be a useful tool for scoring 3D structural models. We used the contacts predicted by the CASP8 residue–residue contact prediction groups to select models for each target domain submitted to the experiment. We found that the information contained in the predicted residue–residue contacts would probably have helped in the selection of 3D models in the free modeling regime and over the harder comparative modeling targets. Indeed, in many cases, the models selected using just the predicted contacts had better GDT‐TS scores than all but the best 3D prediction groups. Despite the well‐known low accuracy of residue–residue contact predictions, it is clear that the predictive power of contacts can be useful in 3D model prediction strategies. Proteins 2010. © 2010 Wiley‐Liss, Inc.     

A method for simultaneous alignment of multiple protein structures

Here, we present MultiProt, a fully automated highly efficient technique to detect multiple structural alignments of protein structures. MultiProt finds the common geometrical cores between input molecules. To date, most methods for multiple alignment start from the pairwise alignment solutions. This may lead to a small overall alignment. In contrast, our method derives multiple alignments from simultaneous superpositions of input molecules. Further, our method does not require that all input molecules participate in the alignment. Actually, it efficiently detects high scoring partial multiple alignments for all possible number of molecules in the input. To demonstrate the power of MultiProt, we provide a number of case studies. First, we demonstrate known multiple alignments of protein structures to illustrate the performance of MultiProt. Next, we present various biological applications. These include: (1) a partial alignment of hinge-bent domains; (2) identification of functional groups of G-proteins; (3) analysis of binding sites; and (4) protein-protein interface alignment. Some applications preserve the sequence order of the residues in the alignment, whereas others are order-independent. It is their residue sequence order-independence that allows application of MultiProt to derive multiple alignments of binding sites and of protein-protein interfaces, making MultiProt an extremely useful structural tool.     

RNA2D3D: A program for Generating,Viewing, and Comparing 3-Dimensional Models of RNA

Abstract

Using primary and secondary structure information of an RNA molecule, the program RNA2D3D automatically and rapidly produces a first-order approximation of a 3-dimensional conformation consistent with this information. Applicable to structures of arbitrary branching complexity and pseudoknot content, it features efficient interactive graphical editing for the removal of any overlaps introduced by the initial generating procedure and for making conformational changes favorable to targeted features and subsequent refinement. With emphasis on fast exploration of alternative 3D conformations, one may interactively add or delete base-pairs, adjacent stems can be coaxially stacked or unstacked, single strands can be shaped to accommodate special constraints, and arbitrary subsets can be defined and manipulated as rigid bodies. Compaction, whereby base stacking within stems is optimally extended into connecting single strands, is also available as a means of strategically making the structures more compact and revealing folding motifs. Subsequent refinement of the first-order approximation, of modifications, and for the imposing of tertiary constraints is assisted with standard energy refinement techniques. Previously determined coordinates for any part of the molecule are readily incorporated, and any part of the modeled structure can be output as a PDB or XYZ file. Illustrative applications in the areas of ribozymes, viral kissing loops, viral internal ribosome entry sites, and nanobiology are presented.     

G‐LoSA: An efficient computational tool for local structure‐centric biological studies and drug design

Molecular recognition by protein mostly occurs in a local region on the protein surface. Thus, an efficient computational method for accurate characterization of protein local structural conservation is necessary to better understand biology and drug design. We present a novel local structure alignment tool, G‐LoSA. G‐LoSA aligns protein local structures in a sequence order independent way and provides a GA‐score, a chemical feature‐based and size‐independent structure similarity score. Our benchmark validation shows the robust performance of G‐LoSA to the local structures of diverse sizes and characteristics, demonstrating its universal applicability to local structure‐centric comparative biology studies. In particular, G‐LoSA is highly effective in detecting conserved local regions on the entire surface of a given protein. In addition, the applications of G‐LoSA to identifying template ligands and predicting ligand and protein binding sites illustrate its strong potential for computer‐aided drug design. We hope that G‐LoSA can be a useful computational method for exploring interesting biological problems through large‐scale comparison of protein local structures and facilitating drug discovery research and development. G‐LoSA is freely available to academic users at http://im.compbio.ku.edu/GLoSA/ .     

RigidFinder: A fast and sensitive method to detect rigid blocks in large macromolecular complexes

Advances in structure determination have made possible the analysis of large macromolecular complexes (some with nearly 10,000 residues, such as GroEL). The large‐scale conformational changes associated with these complexes require new approaches. Historically, a crucial component of motion analysis has been the identification of moving rigid blocks from the comparison of different conformations. However, existing tools do not allow consistent block identification in very large structures. Here, we describe a novel method, RigidFinder, for such identification of rigid blocks from different conformations—across many scales, from large complexes to small loops. RigidFinder defines rigidity in terms of blocks, where inter‐residue distances are conserved across conformations. Distance conservation, unlike the averaged values (e.g., RMSD) used by many other methods, allows for sensitive identification of motions. A further distinguishing feature of our method, is that, it is capable of finding blocks made from nonconsecutive fragments of multiple polypeptide chains. In our implementation, we utilize an efficient quasi‐dynamic programming search algorithm that allows for real‐time application to very large structures. RigidFinder can be used at a dedicated web server ( http://rigidfinder.molmovdb.org ). The server also provides links to examples at various scales such as loop closure, domain motions, partial refolding, and subunit shifts. Moreover, here we describe the detailed application of RigidFinder to four large structures: Pyruvate Phosphate Dikinase, T7 RNA polymerase, RNA polymerase II, and GroEL. The results of the method are in excellent agreement with the expert‐described rigid blocks. Proteins 2010. © 2009 Wiley‐Liss, Inc.     

Discriminating physiological from non-physiological interfaces in structures of protein complexes: A community-wide study

Reliably scoring and ranking candidate models of protein complexes and assigning their oligomeric state from the structure of the crystal lattice represent outstanding challenges. A community-wide effort was launched to tackle these challenges. The latest resources on protein complexes and interfaces were exploited to derive a benchmark dataset consisting of 1677 homodimer protein crystal structures, including a balanced mix of physiological and non-physiological complexes. The non-physiological complexes in the benchmark were selected to bury a similar or larger interface area than their physiological counterparts, making it more difficult for scoring functions to differentiate between them. Next, 252 functions for scoring protein-protein interfaces previously developed by 13 groups were collected and evaluated for their ability to discriminate between physiological and non-physiological complexes. A simple consensus score generated using the best performing score of each of the 13 groups, and a cross-validated Random Forest (RF) classifier were created. Both approaches showed excellent performance, with an area under the Receiver Operating Characteristic (ROC) curve of 0.93 and 0.94, respectively, outperforming individual scores developed by different groups. Additionally, AlphaFold2 engines recalled the physiological dimers with significantly higher accuracy than the non-physiological set, lending support to the reliability of our benchmark dataset annotations. Optimizing the combined power of interface scoring functions and evaluating it on challenging benchmark datasets appears to be a promising strategy.     

Towards a structural classification of phosphate binding sites in protein-nucleotide complexes: an automated all-against-all structural comparison using geometric matching

A method is described for the rapid comparison of protein binding sites using geometric matching to detect similar three-dimensional structure. The geometric matching detects common atomic features through identification of the maximum common sub-graph or clique. These features are not necessarily evident from sequence or from global structural similarity giving additional insight into molecular recognition not evident from current sequence or structural classification schemes. Here we use the method to produce an all-against-all comparison of phosphate binding sites in a number of different nucleotide phosphate-binding proteins. The similarity search is combined with clustering of similar sites to allow a preliminary structural classification. Clustering by site similarity produces a classification of binding sites for the 476 representative local environments producing ten main clusters representing half of the representative environments. The similarities make sense in terms of both structural and functional classification schemes. The ten main clusters represent a very limited number of unique structural binding motifs for phosphate. These are the structural P-loop, di-nucleotide binding motif [FAD/NAD(P)-binding and Rossman-like fold] and FAD-binding motif. Similar classification schemes for nucleotide binding proteins have also been arrived at independently by others using different methods.     

Coevolutionary constraints in the sequence‐space of macromolecular complexes reflect their self‐assembly pathways

Is the order in which biomolecular subunits self‐assemble into functional macromolecular complexes imprinted in their sequence‐space? Here, we demonstrate that the temporal order of macromolecular complex self‐assembly can be efficiently captured using the landscape of residue‐level coevolutionary constraints. This predictive power of coevolutionary constraints is irrespective of the structural, functional, and phylogenetic classification of the complex and of the stoichiometry and quaternary arrangement of the constituent monomers. Combining this result with a number of structural attributes estimated from the crystal structure data, we find indications that stronger coevolutionary constraints at interfaces formed early in the assembly hierarchy probably promotes coordinated fixation of mutations that leads to high‐affinity binding with higher surface area, increased surface complementarity and elevated number of molecular contacts, compared to those that form late in the assembly. Proteins 2017; 85:1183–1189. © 2017 Wiley Periodicals, Inc.     

The novel 1D chain and 3D network of mercury carborane-diphenylphosphine complexes constructed by covalent bond or hydrogen bond interactions

The closo- and nido-carborane-diphenylphosphine complexes [Hg₂{1,2-(PPh₂)₂-1,2-C₂B₁₀H₁₀}₂(μ-Cl₂)₂(μ-HgCl₂)₃]·2CH₂Cl₂ (1) and [HgCl(PPh₃){7,8-(PPh₂)₂-7,8-C₂B₉H₁₀}] (2) have been synthesized and characterized by elemental analysis, FT-IR and X-ray structure determination. The X-ray structure analysis for these two complexes showed that the carborane cage ligand was coordinated bidentately to the Hg(II) center through its two phosphorus atoms. The coordination geometry of the mercury atom complexed by P₂Cl₂ unit in complex 1 or P₃Cl unit in complex 2 was a distorted tetrahedron, while the mercury atom in complex 2 coordinated to six Cl atoms was a slightly distorted octahedron. X-ray analysis reveals that the complex 1 forms a 1D chain coordination polymer via bridged Hg-Cl bonds. For complex 2, it displays a 3D network constructed by the C-H···Cl hydrogen bonds and C-H···H-B dihydrogen bonds.     

The Bio3D packages for structural bioinformatics

Bio3D is a family of R packages for the analysis of biomolecular sequence, structure, and dynamics. Major functionality includes biomolecular database searching and retrieval, sequence and structure conservation analysis, ensemble normal mode analysis, protein structure and correlation network analysis, principal component, and related multivariate analysis methods. Here, we review recent package developments, including a new underlying segregation into separate packages for distinct analysis, and introduce a new method for structure analysis named ensemble difference distance matrix analysis (eDDM). The eDDM approach calculates and compares atomic distance matrices across large sets of homologous atomic structures to help identify the residue wise determinants underlying specific functional processes. An eDDM workflow is detailed along with an example application to a large protein family. As a new member of the Bio3D family, the Bio3D‐eddm package supports both experimental and theoretical simulation‐generated structures, is integrated with other methods for dissecting sequence‐structure–function relationships, and can be used in a highly automated and reproducible manner. Bio3D is distributed as an integrated set of platform independent open source R packages available from: http://thegrantlab.org/bio3d/ .     

CRK: An evolutionary approach for distinguishing biologically relevant interfaces from crystal contacts

Protein crystals contain two different types of interfaces: biologically relevant ones, observed in protein–protein complexes and oligomeric proteins, and nonspecific ones, corresponding to crystal lattice contacts. Because of the increasing complexity of the objects being tackled in structural biology, distinguishing biological contacts from crystal contacts is not always a trivial task and can lead to wrong interpretation of macromolecular structures. We devised an approach (CRK, core‐rim K_a/K_s ratio) for distinguishing biologically relevant interfaces from nonspecific ones. Given a protein–protein interface, CRK finds a set of homologs to the sequences of the proteins involved in the interface, retrieves and aligns the corresponding coding sequences, on which it carries out a residue‐by‐residue K_a/K_s ratio (ω) calculation. It divides interface residues into a “rim” and a “core” set and analyzes the selection pressure on the residues belonging to the two sets. We developed and tested CRK on different datasets and test cases, consisting of biologically relevant contacts, nonspecific ones or of both types. The method proves very effective in distinguishing the two categories of interfaces, with an overall accuracy rate of 84%. As it relies on different principles when compared with existing tools, CRK is optimally suited to be used in combination with them. In addition, CRK has potential applications in the validation of structures of oligomeric proteins and protein complexes. Proteins 2010. © 2010 Wiley‐Liss, Inc.     

Sequence-structure-function relationships of a tRNA (m7G46) methyltransferase studied by homology modeling and site-directed mutagenesis

The Escherichia coli TrmB protein and its Saccharomyces cerevisiae ortholog Trm8p catalyze the S-adenosyl-L-methionine-dependent formation of 7-methylguanosine at position 46 (m7G46) in tRNA. To learn more about the sequence-structure-function relationships of these enzymes we carried out a thorough bioinformatics analysis of the tRNA:m7G methyltransferase (MTase) family to predict sequence regions and individual amino acid residues that may be important for the interactions between the MTase and the tRNA substrate, in particular the target guanosine 46. We used site-directed mutagenesis to construct a series of alanine substitutions and tested the activity of the mutants to elucidate the catalytic and tRNA-recognition mechanism of TrmB. The functional analysis of the mutants, together with the homology model of the TrmB structure and the results of the phylogenetic analysis, revealed the crucial residues for the formation of the substrate-binding site and the catalytic center in tRNA:m7G MTases.     

Recognition of gluconeogenic enzymes; Icl1, Fbp1, and Mdh2 by Gid4 ligase: A molecular docking study

The pro/N‐degron pathway is an evolved protein degradation pathway through the ubiquitin‐proteasome system. It is a vital pathway to attain protein homeostasis inside the liver cells with varying glucose levels. N‐terminal proline exists in more than 300 proteins in Saccharomyces cerevisiae, but only three of them are the gluconeogenic enzymes; isocitrate lyase (Icl1), fructose‐1,6‐bisphosphatase (Fbp1), and malate dehydrogenase (Mdh2). The present in silico study aims to structurally illustrate the binding of Icl1 enzyme to Gid4 ligase concerning its peers; Fbp1 and Mdh2. Based on the molecular docking scores and interactions, one can attribute the binding stability of Gid4 with degrons, to peptides of length six up to eight from the N‐terminal. Moreover, the percent change in the docking score provides a rationale for the unique Gid4‐Icl1^1‐4 interaction. The present study provides insights on the binding attitude of Gid4 ligase to degrons of different lengths, so one will consider in designing peptidomimetics to target Gid4 ligase.     

Evolutionarily conserved regions and hydrophobic contacts at the superfamily level: The case of the fold-type I, pyridoxal-5'-phosphate-dependent enzymes

The wealth of biological information provided by structural and genomic projects opens new prospects of understanding life and evolution at the molecular level. In this work, it is shown how computational approaches can be exploited to pinpoint protein structural features that remain invariant upon long evolutionary periods in the fold-type I, PLP-dependent enzymes. A nonredundant set of 23 superposed crystallographic structures belonging to this superfamily was built. Members of this family typically display high-structural conservation despite low-sequence identity. For each structure, a multiple-sequence alignment of orthologous sequences was obtained, and the 23 alignments were merged using the structural information to obtain a comprehensive multiple alignment of 921 sequences of fold-type I enzymes. The structurally conserved regions (SCRs), the evolutionarily conserved residues, and the conserved hydrophobic contacts (CHCs) were extracted from this data set, using both sequence and structural information. The results of this study identified a structural pattern of hydrophobic contacts shared by all of the superfamily members of fold-type I enzymes and involved in native interactions. This profile highlights the presence of a nucleus for this fold, in which residues participating in the most conserved native interactions exhibit preferential evolutionary conservation, that correlates significantly (r = 0.70) with the extent of mean hydrophobic contact value of their apolar fraction.     

The role of geometric complementarity in secondary structure packing: a systematic docking study

A strong similarity between the major aspects of protein folding and protein recognition is one of the emerging fundamental principles in protein science. A crucial importance of steric complementarity in protein recognition is a well-established fact. The goal of this study was to assess the importance of the steric complementarity in protein folding, namely, in the packing of the secondary structure elements. Although the tight packing of protein structures, in general, is a well-known fact, a systematic study of the role of geometric complementarity in the packing of secondary structure elements has been lacking. To assess the role of the steric complementarity, we used a docking procedure to recreate the crystallographically determined packing of secondary structure elements in known protein structures by using the geometric match only. The docking results revealed a significant percentage of correctly predicted packing configurations. Different types of pairs of secondary structure elements showed different degrees of steric complementarity (from high to low: beta-beta, loop-loop, alpha-alpha, and alpha-beta). Interestingly, the relative contribution of the steric match in different types of pairs was correlated with the number of such pairs in known protein structures. This effect may indicate an evolutionary pressure to select tightly packed elements of secondary structure to maximize the packing of the entire structure. The overall conclusion is that the steric match plays an essential role in the packing of secondary structure elements. The results are important for better understanding of principles of protein structure and may facilitate development of better methods for protein structure prediction.     

Taking advantage of local structure descriptors to analyze interresidue contacts in protein structures and protein complexes

Interresidue protein contacts in proteins structures and at protein-protein interface are classically described by the amino acid types of interacting residues and the local structural context of the contact, if any, is described using secondary structures. In this study, we present an alternate analysis of interresidue contact using local structures defined by the structural alphabet introduced by Camproux et al. This structural alphabet allows to describe a 3D structure as a sequence of prototype fragments called structural letters, of 27 different types. Each residue can then be assigned to a particular local structure, even in loop regions. The analysis of interresidue contacts within protein structures defined using Vorono? tessellations reveals that pairwise contact specificity is greater in terms of structural letters than amino acids. Using a simple heuristic based on specificity score comparison, we find that 74% of the long-range contacts within protein structures are better described using structural letters than amino acid types. The investigation is extended to a set of protein-protein complexes, showing that the similar global rules apply as for intraprotein contacts, with 64% of the interprotein contacts best described by local structures. We then present an evaluation of pairing functions integrating structural letters to decoy scoring and show that some complexes could benefit from the use of structural letter-based pairing functions.     

Ter-Seq: A high-throughput method to stabilize transient ternary complexes and measure associated kinetics

Regulation of biological processes by proteins often involves the formation of transient, multimeric complexes whose characterization is mechanistically important but challenging. The bacterial toxin CcdB binds and poisons DNA Gyrase. The corresponding antitoxin CcdA extracts CcdB from its complex with Gyrase through the formation of a transient ternary complex, thus rejuvenating Gyrase. We describe a high throughput methodology called Ter-Seq to stabilize probable ternary complexes and measure associated kinetics using the CcdA-CcdB-GyrA14 ternary complex as a model system. The method involves screening a yeast surface display (YSD) saturation mutagenesis library of one partner (CcdB) for mutants that show enhanced ternary complex formation. We also isolated CcdB mutants that were either resistant or sensitive to rejuvenation, and used surface plasmon resonance (SPR) with purified proteins to validate the kinetics measured using the surface display. Positions, where CcdB mutations lead to slower rejuvenation rates, are largely involved in CcdA-binding, though there were several notable exceptions suggesting allostery. Mutations at these positions reduce the affinity towards CcdA, thereby slowing down the rejuvenation process. Mutations at GyrA14-interacting positions significantly enhanced rejuvenation rates, either due to reduced affinity or complete loss of CcdB binding to GyrA14. We examined the effect of different parameters (CcdA affinity, GyrA14 affinity, surface accessibilities, evolutionary conservation) on the rate of rejuvenation. Finally, we further validated the Ter-Seq results by monitoring the kinetics of ternary complex formation for individual CcdB mutants in solution by fluorescence resonance energy transfer (FRET) studies.     

细菌脲酶蛋白复合物及其活化机制

脲酶能够催化尿素分解生成氨,在农业和医学领域中具有重要的意义。细菌脲酶蛋白包括结构蛋白(UreA、UreB和UreC)和辅助蛋白(UreD/UreH、UreE、UreF和UreG),它们在脲酶活化过程中各自具有独特的作用,结构蛋白形成脲酶活性中心,而辅助蛋白主要负责镍离子的传递。文中综述了细菌脲酶蛋白复合物的结构和功能,以及各蛋白之间如何相互作用完成其活化过程,以期为脲酶活性调控研究及脲酶抑制剂开发等提供理论指导。