Catalytic mechanism of α-phosphate attack in dUTPase is revealed by X-ray crystallographic snapshots of distinct intermediates, 31P-NMR spectroscopy and reaction path modelling.

Enzymatic synthesis and hydrolysis of nucleoside phosphate compounds play a key role in various biological pathways, like signal transduction, DNA synthesis and metabolism. Although these processes have been studied extensively, numerous key issues regarding the chemical pathway and atomic movements remain open for many enzymatic reactions. Here, using the Mason-Pfizer monkey retrovirus dUTPase, we study the dUTPase-catalyzed hydrolysis of dUTP, an incorrect DNA building block, to elaborate the mechanistic details at high resolution. Combining mass spectrometry analysis of the dUTPase-catalyzed reaction carried out in and quantum mechanics/molecular mechanics (QM/MM) simulation, we show that the nucleophilic attack occurs at the α-phosphate site. Phosphorus-31 NMR spectroscopy ((31)P-NMR) analysis confirms the site of attack and shows the capability of dUTPase to cleave the dUTP analogue α,ß-imido-dUTP, containing the imido linkage usually regarded to be non-hydrolyzable. We present numerous X-ray crystal structures of distinct dUTPase and nucleoside phosphate complexes, which report on the progress of the chemical reaction along the reaction coordinate. The presently used combination of diverse structural methods reveals details of the nucleophilic attack and identifies a novel enzyme-product complex structure.

Discovery of novel MDR-Mycobacterium tuberculosis inhibitor by new FRIGATE computational screen.

With 1.6 million casualties annually and 2 billion people being infected, tuberculosis is still one of the most pressing healthcare challenges. Here we report on the new computational docking algorithm FRIGATE which unites continuous local optimization techniques (conjugate gradient method) with an inherently discrete computational approach in forcefield computation, resulting in equal or better scoring accuracies than several benchmark docking programs. By utilizing FRIGATE for a virtual screen of the ZINC library against the Mycobacterium tuberculosis (Mtb) enzyme antigen 85C, we identified novel small molecule inhibitors of multiple drug-resistant Mtb, which bind in vitro to the catalytic site of antigen 85C.

A hybrid clustering of protein binding sites.

The Protein Data Bank contains the description of approximately 27 000 protein-ligand binding sites. Most of the ligands at these sites are biologically active small molecules, affecting the biological function of the protein. The classification of their binding sites may lead to relevant results in drug discovery and design. Clusters of similar binding sites were created here by a hybrid, sequence and spatial structure-based approach, using the OPTICS clustering algorithm. A dissimilarity measure was defined: a distance function on the amino acid sequences of the binding sites. All the binding sites were clustered in the Protein Data Bank according to this distance function, and it was found that the clusters characterized well the Enzyme Commission numbers of the entries. The results, carefully color coded by the Enzyme Commission numbers of the proteins, containing the 20 967 binding sites clustered, are available as html files in three parts at http://pitgroup.org/seqclust/.

Cysteine and tryptophan anomalies found when scanning all the binding sites in the Protein Data Bank.

The Protein Data Bank (PDB) is one of the richest sources of structural biological information in the World. It started to exist as the computer-readable depository of crystallographic data complementing printed papers. The proper interpretation of the content of the individual files in the PDB still needs the detailed information found in the citing publication. An advanced graph theoretical method is presented here for automatically repairing, re-organising and re-structuring PDB data yielding the identification of all the protein-ligand complexes and all the binding sites in the PDB. As an application, we identified strong cysteine and tryptophan irregularities in the data.

On the asymmetry of the residue compositions of the binding sites on protein surfaces.

By screening all the ligand binding sites in the Protein Data Bank, we have found that while it is geometrically possible that a loop, formed from a protein chain with residues ZYX, would "impersonate" another chain-loop with residues XYZ by a simple twisting of either the loop or the bound ligand, it almost never happens. This fact is rather surprising, and implies a notable asymmetry, since (i) loops in the folded proteins sometimes can be flexible enough to be twisted, but (ii) ligands are almost always extremely mobile before binding to the protein, therefore they can turn around and bind to residue-sequence ZYX as well. Data availability: The supplementary Table 3 lists the appearances of the residue-sequences and their inverses in the binding sites of the whole PDB, and is available at http://www.worldscient.com/jbcb/.

DECOMP: a PDB decomposition tool on the web.

The protein databank (PDB) contains high quality structural data for computational structural biology investigations. We have earlier described a fast tool (the decomp_pdb tool) for identifying and marking missing atoms and residues in PDB files. The tool also automatically decomposes PDB entries into separate files describing ligands and polypeptide chains. Here, we describe a web interface named DECOMP for the tool. Our program correctly identifies multi-monomer ligands, and the server also offers the preprocessed ligand-protein decomposition of the complete PDB for downloading (up to size: 5GB) AVAILABILITY: http://decomp.pitgroup.org.

Four spatial points that define enzyme families.

The catalytic properties of enzymes, containing the Asp-His-Ser triads are deeply investigated for a long time. Serine endopeptidases, cutinases, acetylcholinesterases, cellulases, among other enzymes, contain these triads. We found that solely the geometric properties of just four points in the spatial structure of these enzymes are characteristic to their family.

Analyzing the simplicial decomposition of spatial protein structures.

BACKGROUND: The fast growing Protein Data Bank contains the three-dimensional description of more than 45000 protein- and nucleic-acid structures today. The large majority of the data in the PDB are measured by X-ray crystallography by thousands of researchers in millions of work-hours. Unfortunately, lots of structural errors, bad labels, missing atoms, falsely identified chains and groups make dificult the automated processing of this treasury of structural biological data. RESULTS: After we performed a rigorous re-structuring of the whole PDB on graph-theoretical basis, we created the RS-PDB (Rich-Structure PDB) database. Using this cleaned and repaired database, we defined simplicial complexes on the heavy-atoms of the PDB, and analyzed the tetrahedra for geometric properties. CONCLUSION: We have found surprisingly characteristic differences between simplices with atomic vertices of different types, and between the atomic neighborhoods--described also by simplices--of different ligand atoms in proteins.

High throughput processing of the structural information in the protein data bank.

The protein data bank (PDB) is the largest, most comprehensive, freely available depository of protein structural information, containing more than 37,500 deposited structures. On one hand, the form and the organization of the PDB seems to be perfectly adequate for gathering information from specific protein structures, by using the bibliographic references and the informative remark fields. On the other hand, however, it seems to be impossible to automatically review remark fields and journal references for processing hundreds or thousands of PDB files. We present here a family of combinatorial algorithms to solve some of these problems. Our algorithms are capable to automatically analyze PDB structural information, identify missing atoms, repair chain ID information, and most importantly, the algorithms are capable of identifying ligands with their respective binding sites.

Being a binding site: characterizing residue composition of binding sites on proteins.

The Protein Data Bank contains the description of more than 45,000 three-dimensional protein and nucleic-acid structures today. Started to exist as the computer-readable depository of crystallographic data complementing printed articles, the proper interpretation of the content of the individual files in the PDB still frequently needs the detailed information found in the citing publication. This fact implies that the fully automatic processing of the whole PDB is a very hard task. We first cleaned and re-structured the PDB data, then analyzed the residue composition of the binding sites in the whole PDB for frequency and for hidden association rules. Main results of the paper: (i) the cleaning and repairing algorithm (ii) redundancy elimination from the data (iii) application of association rule mining to the cleaned non-redundant data set. We have found numerous significant relations of the residue-composition of the ligand binding sites on protein surfaces, summarized in two figures. One of the classical data-mining methods for exploring implication-rules, the association-rule mining, is capable to find previously unknown residue-set preferences of bind ligands on protein surfaces. Since protein-ligand binding is a key step in enzymatic mechanisms and in drug discovery, these uncovered preferences in the study of more than 19,500 binding sites may help in identifying new binding protein-ligand pairs.

Building a structured PDB: the RS-PDB database.

A method for automatically analyzing structures deposited in the Protein Data Bank is presented. The method is capable to detect missing atoms, bond length deviations, atom bumps and to correctly identify protein-ligand complexes. The results are organized into a database, called the Rich Structure PDB (RS-PDB in short) from which one can easily select PDB entries satisfying diverse sets of requirements. The newer and richer mmCIF format of both the PDB and its chemical component dictionary (formerly the HET Group Dictionary) were used in the construction, and the International Chemical Identifier (InChI) of IUPAC played a main role in correctly identifying distinct ligands.