Analysis of crystal structures of aspartic proteinases: On the role of amino acid residues adjacent to the catalytic site of pepsin-like enzymes

To elucidate the role of amino acid residues adjacent to the catalytic site of pepsin-like enzymes, we analyzed and compared the crystal structures of these enzymes, their complexes with inhibitors, and zymogens in the active site area (a total of 82 structures). In addition to the water molecule (W1) located between the active carboxyls and playing a role of the nucleophile during catalytic reaction, another water molecule (W2) at the vicinity of the active groups was found to be completely conserved. This water molecule plays an essential role in formation of a chain of hydrogen-bonded residues between the active site flap and the active carboxyls on ligand binding. These data suggest a new approach to understanding the role of residues around the catalytic site, which can assist the development of the catalytic reaction. The influence of groups adjacent to the active carboxyls is manifested by pepsin activity at pH 1.0. Some features of pepsin-like enzymes and their mutants are discussed in the framework of the approach.     

Modeling of substrate and inhibitory complexes of histidine-aspartic protease

A three-dimensional structure of histo-aspartic protease (HAP), a pepsin-like enzyme from the causative agent of malaria Plasmodium falciparum, is suggested on the basis of homologous modeling followed by equilibration by the method of molecular dynamics. The presence of a His residue in the catalytic site instead of an Asp residue, which is characteristic of pepsin-like enzymes, and replacement of some other conserved residues in the active site make it possible for the enzyme to function by the covalent mechanism inherent in serine proteases. The detailed structures of HAP complexes with pepstatin, a noncovalent inhibitor of aspartic proteases, and phenylmethylsulfonyl fluoride, a covalent inhibitor of serine proteases, as well as with a pentapeptide substrate are discussed.     

Conserved Interactions of the Active Carboxyls in Pepsin-like Enzymes and Retroviral Proteases

In addition to previous studies, 30 crystal structures of retroviral proteases corresponding to the highest resolution were inspected to analyze the interactions of the active carboxyls with surroundings groups. The outer oxygens of the active carboxyls in retroviral enzymes form contacts only with the water molecule participating in catalysis. This is an important difference between retroviral proteases and pepsin-like enzymes, which form a net of hydrogen bonds of these outer oxygens with residues neighboring the catalytic site in 3D structures. At the same time, it was found that in all aspartic proteases the inner oxygens of the active carboxyls are also involved in the chain of interactions through peptide groups Thr–Gly adjacent to the active residues. Polarization of these peptide groups influences the donor–acceptor properties of the active carboxyls. The found chain of interactions is more extensive in retroviral than in pepsin-like proteases; however, its main part is conserved for the whole class of these enzymes. Some implications of the role of these interactions are discussed.     

Conserved interactions of the active carboxyls in pepsin-like enzymes and retroviral proteases

In addition to previous studies, 30 crystal structures of retroviral proteases corresponding to the highest resolution were inspected to analyze the interactions of the active carboxyl with surroundings groups. The outer oxygen of the active carboxyl in retroviral enzymes form contacts only with the water molecule participating in catalysis. This is an important difference between retroviral proteases and pepsin-like enzymes, which form a net of hydrogen bonds of these outer oxygen with residues neighboring the catalytic site in 3D structures. At the same time, it was found that in all aspartic proteases the inner oxygen of the active carboxyl are also involved in the chain of interactions through peptide groups Thr-Gly adjacent to the active residues. Polarization of these peptide groups influences the donor-acceptor properties of the active carboxyl. The found chain of interactions is more extensive in retroviral than in pepsin-like proteases; however, its main part is conserved for the whole class of these enzymes. Some implications of the role of these interactions are discussed.     

Modeling of substrate and inhibitory complexes of histidine-aspartic protease

A three-dimensional structure of histo-aspartic protease (HAP), a pepsin-like enzyme from the causative agent of malaria Plasmodium falciparum, is suggested on the basis of homologous modeling followed by equilibration by the method of molecular dynamics. The presence of a His residue in the catalytic site instead of an Asp residue, which is characteristic of pepsin-like enzymes, and replacement of some other conserved residues in the active site make it possible for the enzyme to function by the covalent mechanism inherent in serine proteases. The detailed structures of HAP complexes with pepstatin, a noncovalent inhibitor of aspartic proteases, and phenylmethylsulfonyl fluoride, a covalent inhibitor of serine proteases, as well as with a pentapeptide substrate are discussed.     

Acyltransferase families that act on thioesters: Sequences,structures, and mechanisms

Acyltransferases (AT) are enzymes that catalyze the transfer of acyl group to a receptor molecule. This review focuses on ATs that act on thioester-containing substrates. Although many ATs can recognize a wide variety of substrates, sequence similarity analysis allowed us to classify the ATs into fifteen distinct families. Each AT family is originated from enzymes experimentally characterized to have AT activity, classified according to sequence similarity, and confirmed with tertiary structure similarity for families that have crystallized structures available. All the sequences and structures of the AT families described here are present in the thioester-active enzyme (ThYme) database. The AT sequences and structures classified into families and available in the ThYme database could contribute to enlightening the understanding acyl transfer to thioester-containing substrates, most commonly coenzyme A, which occur in multiple metabolic pathways, mostly with fatty acids.     

Studies on the proteolytic enzymes of invertebrates constituting fish food

Studies were carried out on the proteolytic activity of trypsin and pepsin-like enzymes of invertebrates consituting fish food. Relations between proteolytic activity of enzymes, pH, and temperature were established.     

Evaluating protein structures determined by structural genomics consortia

Structural genomics projects are providing large quantities of new 3D structural data for proteins. To monitor the quality of these data, we have developed the protein structure validation software suite (PSVS), for assessment of protein structures generated by NMR or X-ray crystallographic methods. PSVS is broadly applicable for structure quality assessment in structural biology projects. The software integrates under a single interface analyses from several widely-used structure quality evaluation tools, including PROCHECK (Laskowski et al., J Appl Crystallog 1993;26:283-291), MolProbity (Lovell et al., Proteins 2003;50:437-450), Verify3D (Luthy et al., Nature 1992;356:83-85), ProsaII (Sippl, Proteins 1993;17: 355-362), the PDB validation software, and various structure-validation tools developed in our own laboratory. PSVS provides standard constraint analyses, statistics on goodness-of-fit between structures and experimental data, and knowledge-based structure quality scores in standardized format suitable for database integration. The analysis provides both global and site-specific measures of protein structure quality. Global quality measures are reported as Z scores, based on calibration with a set of high-resolution X-ray crystal structures. PSVS is particularly useful in assessing protein structures determined by NMR methods, but is also valuable for assessing X-ray crystal structures or homology models. Using these tools, we assessed protein structures generated by the Northeast Structural Genomics Consortium and other international structural genomics projects, over a 5-year period. Protein structures produced from structural genomics projects exhibit quality score distributions similar to those of structures produced in traditional structural biology projects during the same time period. However, while some NMR structures have structure quality scores similar to those seen in higher-resolution X-ray crystal structures, the majority of NMR structures have lower scores. Potential reasons for this "structure quality score gap" between NMR and X-ray crystal structures are discussed.     

Empirical isotropic chemical shift surfaces

A list of proteins is given for which spatial structures, with a resolution better than 2.5 A, are known from entries in the Protein Data Bank (PDB) and isotropic chemical shift (ICS) values are known from the RefDB database related to the Biological Magnetic Resonance Bank (BMRB) database. The structures chosen provide, with unknown uncertainties, dihedral angles phi and psi characterizing the backbone structure of the residues. The joint use of experimental ICSs of the same residues within the proteins, again with mostly unknown uncertainties, and ab initio ICS(phi,psi) surfaces obtained for the model peptides For-(L-Ala)(n)-NH(2), with n = 1, 3, and 5, resulted in so-called empirical ICS(phi,psi) surfaces for all major nuclei of the 20 naturally occurring alpha-amino acids. Out of the many empirical surfaces determined, it is the 13C(alpha)-1H(alpha) ICS(phi,psi) surface which seems to be most promising for identifying major secondary structure types, alpha-helix, beta-strand, left-handed helix (alpha(D)), and polyproline-II. Detailed tests suggest that Ala is a good model for many naturally occurring alpha-amino acids. Two-dimensional empirical 13C(alpha)-1H(alpha) ICS(phi,psi) correlation plots, obtained so far only from computations on small peptide models, suggest the utility of the experimental information contained therein and thus they should provide useful constraints for structure determinations of proteins.     

An automatic method involving cluster analysis of secondary structures for the identification of domains in proteins.

With a growing number of structures available in the Brookhaven Protein Data Bank, automatic methods for domain identification are required for the construction of databases. Domains are considered to be clusters of secondary structure elements. Thus, helices and strands are first clustered using intersecondary structural distances between C alpha positions, and dendrograms based on this distance measure are used to identify domains. Individual domains are recognized by a disjoint factor, which enables the automatic identification and classification into disjoint, interacting, and conjoint domains. Application to a database of 83 protein families and 18 unique structures shows that the approach provides an effective delineation of boundaries and identifies those proteins that can be considered as a single domain. A quantitative estimate of the interaction between domains has been proposed. The database of protein domains is a useful tool for understanding protein folding, for recognizing protein folds, and for understanding structure-activity relationships.     

TESS: a geometric hashing algorithm for deriving 3D coordinate templates for searching structural databases. Application to enzyme active sites.

It is well established that sequence templates such as those in the PROSITE and PRINTS databases are powerful tools for predicting the biological function and tertiary structure for newly derived protein sequences. The number of X-ray and NMR protein structures is increasing rapidly and it is apparent that a 3D equivalent of the sequence templates is needed. Here, we describe an algorithm called TESS that automatically derives 3D templates from structures deposited in the Brookhaven Protein Data Bank. While a new sequence can be searched for sequence patterns, a new structure can be scanned against these 3D templates to identify functional sites. As examples, 3D templates are derived for enzymes with an O-His-O "catalytic triad" and for the ribonucleases and lysozymes. When these 3D templates are applied to a large data set of nonidentical proteins, several interesting hits are located. This suggests that the development of a 3D template database may help to identify the function of new protein structures, if unknown, as well as to design proteins with specific functions.     

A new residue-nucleotide propensity potential with structural information considered for discriminating protein-RNA docking decoys

Understanding the key factors that influence the preferences of residue-nucleotide interactions in specific protein-RNA interactions has remained a research focus. We propose an effective approach to derive residue-nucleotide propensity potentials through considering both the types of residues and nucleotides, and secondary structure information of proteins and RNAs from the currently largest nonredundant and nonribosomal protein-RNA interaction database. To test the validity of the potentials, we used them to select near-native structures from protein-RNA docking poses. The results show that considering secondary structure information, especially for RNAs, greatly improves the predictive power of pair potentials. The success rate is raised from 50.7 to 65.5% for the top 2000 structures, and the number of cases in which a near-native structure is ranked in top 50 is increased from 7 to 13 out of 17 cases. Furthermore, the exclusion of ribosomes from the database contributes 8.3% to the success rate. In addition, some very interesting findings follow: (i) the protein secondary structure element π-helix is strongly associated with RNA-binding sites; (ii) the nucleotide uracil occurs frequently in the most preferred pairs in which the unpaired and non-Watson-Crick paired uracils are predominant, which is probably significant in evolution. The new residue-nucleotide potentials can be helpful for the progress of protein-RNA docking methods, and for understanding the mechanisms of protein-RNA interactions.     

Interdomain interactions in aspartic proteases of higher organisms and their analogs in retroviral enzymes

On recent evidence, the efficiency of catalysis and the specificity of aspartic proteases depend considerably on the dynamic properties of particular molecular regions and their correlations. Analysis of the three-dimensional structures of these enzymes showed the presence of a continuous chain of hydrogen-bonded groups, which connects regions with highly correlated dynamic parameters and provides for a “cross-hand” interaction between domains. This chain includes the inner oxygens of the active carboxyls and the conserved internal water molecules. The so-called “fireman grip” interdomain hydrogen bonding is part of this chain. Such networks are abortive in retroviral proteases. The role of these interactions in the functions of aspartic proteases is discussed.     

Functional plasticity in the substrate binding site of beta-secretase

The aspartic protease beta-secretase (BACE) cleaves the amyloid precursor protein into a 42 residue beta-peptide, which is the principal biochemical marker of Alzheimer's disease. Multiple explicit-water molecular dynamics simulations of the apo and inhibitor bound structures of BACE indicate that both open- and closed-flap conformations are accessible at room temperature and should be taken into account for inhibitor design. Correlated motion is observed within each of the two lobes of BACE, as well as for the interfacial region. A self-inhibited conformation with the side chain of Tyr71 occupying the S(1) pocket is present in some of the unbound simulations. The reversible loss of the side chain hydrogen bond between the catalytic Asp32 and Ser35, due to the concomitant reorientation of the Ser35 hydroxyl group and a water molecule conserved in pepsin-like enzymes, provides further evidence for the suggestion that Ser35 assists in proton acceptance and release by Asp32 during catalysis.     

The Search for the Spatial and Electronic Requirements of a Drug

A new program called GAMMA (genetic algorithm for multiple molecule alignment) has been developed for the superimposition of several three-dimensional chemical structures. Superimposition of molecules and evaluation of structural similarity is an important task in drug design and pharmaceutical research. Similarities of compounds are determined by this program either based on their structural or their physicochemical properties by defining different matching criteria. These matching criteria are atomic properties such as atomic number or partial atomic charges. The program is based on a combination of a genetic algorithm with a numerical optimization process. A major goal of this hybrid procedure is to address the conformational flexibility of ligand molecules adequately. Thus, only one conformation per structure is necessary and the program can work even when only one conformation of a compound is stored in a database. The genetic algorithm optimizes in a nondeterministic process the size and the geometric fit of the overlay. The geometric fit of the conformations is further improved by changing torsional angles combining the genetic algorithm and the directed tweak method. The determination of the fitness of a superimposition is based on the Pareto optimization. As an application the superimposition of a set of Cytochrome P450c17 enzyme inhibitors has been performed.Electronic Supplementary Material available.     

Three-dimensional, sequence order-independent structural comparison of a serine protease against the crystallographic database reveals active site similarities: potential implications to evolution and to protein folding.

We have recently developed a fast approach to comparisons of 3-dimensional structures. Our method is unique, treating protein structures as collections of unconnected points (atoms) in space. It is completely independent of the amino acid sequence order. It is unconstrained by insertions, deletions, and chain directionality. It matches single, isolated amino acids between 2 different structures strictly by their spatial positioning regardless of their relative sequential position in the amino acid chain. It automatically detects a recurring 3D motif in protein molecules. No predefinition of the motif is required. The motif can be either in the interior of the proteins or on their surfaces. In this work, we describe an enhancement over our previously developed technique, which considerably reduces the complexity of the algorithm. This results in an extremely fast technique. A typical pairwise comparison of 2 protein molecules requires less than 3 s on a workstation. We have scanned the structural database with dozens of probes, successfully detecting structures that are similar to the probe. To illustrate the power of this method, we compare the structure of a trypsin-like serine protease against the structural database. Besides detecting homologous trypsin-like proteases, we automatically obtain 3D, sequence order-independent, active-site similarities with subtilisin-like and sulfhydryl proteases. These similarities equivalence isolated residues, not conserving the linear order of the amino acids in the chains. The active-site similarities are well known and have been detected by manually inspecting the structures in a time-consuming, laborious procedure. This is the first time such equivalences are obtained automatically from the comparison of full structures. The far-reaching advantages and the implications of our novel algorithm to studies of protein folding, to evolution, and to searches for pharmacophoric patterns are discussed.     

Automated classification of RNA 3D motifs and the RNA 3D Motif Atlas

The analysis of atomic-resolution RNA three-dimensional (3D) structures reveals that many internal and hairpin loops are modular, recurrent, and structured by conserved non-Watson–Crick base pairs. Structurally similar loops define RNA 3D motifs that are conserved in homologous RNA molecules, but can also occur at nonhomologous sites in diverse RNAs, and which often vary in sequence. To further our understanding of RNA motif structure and sequence variability and to provide a useful resource for structure modeling and prediction, we present a new method for automated classification of internal and hairpin loop RNA 3D motifs and a new online database called the RNA 3D Motif Atlas. To classify the motif instances, a representative set of internal and hairpin loops is automatically extracted from a nonredundant list of RNA-containing PDB files. Their structures are compared geometrically, all-against-all, using the FR3D program suite. The loops are clustered into motif groups, taking into account geometric similarity and structural annotations and making allowance for a variable number of bulged bases. The automated procedure that we have implemented identifies all hairpin and internal loop motifs previously described in the literature. All motif instances and motif groups are assigned unique and stable identifiers and are made available in the RNA 3D Motif Atlas (http://rna.bgsu.edu/motifs), which is automatically updated every four weeks. The RNA 3D Motif Atlas provides an interactive user interface for exploring motif diversity and tools for programmatic data access.     

BANMOKI: a searchable database of homology-based 3D models and their electrostatic properties of five bacterial nucleoside monophosphate kinase families

The nucleoside monophosphate kinases (NMPK) are important enzymes that control the ratio of mono- and di-phosphate nucleosides and participate in gene regulation and signal transduction in the cell. However, despite their importance only several 3D structures were experimentally determined in contrast to the wealth of sequences available for each of the NMPK families. To fill this gap we present a Web-based database containing structural models for all proteins of the five bacterial nucleoside monophosphate kinase (bNMPK) families. The models were computed by means of homology-based approach using a few experimentally determined bNMPK structures. The database also contains pK(a) values and their components calculated for the homology-based 3D models, which is a unique feature of the database. The BActerial Nucleoside MOnophosphate KInases (BANMOKI) database is freely accessible (http://www.ces.clemson.edu/compbio/banmoki) and offers an easy user-friendly interface for browsing, searching and downloading content of the database. The users can investigate, using the searching tools of the database, the properties of the bNMP kinases in respect to sequence composition, electrostatic interactions and structural differences.