Relationship between drug structure and minimal inhibitory concentration value used for acceptable daily intake analysis.

The minimal inhibitory concentration (MIC) of an antimicrobial agent for a microbial population (MIC(50, obs) and MIC(90, obs)) is an interpolated value determined for antibacterial drugs by in vitro methods. Many studies have tried to determine the correlation between the MIC(50, obs) or MIC(90, obs) value and the physicochemical parameters to allow quantitaive structure activity relationship (QSAR) predictions of efficacy. A rigorous evaluation of approaches to this problem is presented here. In order to find a correlation between chemical structure and the derivatives of the MIC values for 9 indicatory bacterial strains, it is necessary to employ a number of physicochemical parameters in combination. Only an arithmetic expression composed of many features illustrating the chemical structure of the molecule can be linked to the ƒMIC(50, obs) value. This article demonstrated that, despite the complexity of the MIC value used as the end point, it is possible to validate the model in a limited extent.

Ligand-optimized homology models of D1 and D2 dopamine receptors: application for virtual screening.

Recent breakthroughs in crystallographic studies of G protein-coupled receptors (GPCRs), together with continuous progress in molecular modeling methods, have opened new perspectives for structure-based drug discovery. A crucial enhancement in this area was development of induced fit docking procedures that allow optimization of binding pocket conformation guided by the features of its active ligands. In the course of our research program aimed at discovery of novel antipsychotic agents, our attention focused on dopaminergic D2 and D1 receptors (D2R and D1R). Thus, we decided to investigate whether the availability of a novel structure of the closely related D3 receptor and application of induced fit docking procedures for binding pocket refinement would permit the building of models of D2R and D1R that facilitate a successful virtual screening (VS). Here, we provide an in-depth description of the modeling procedure and the discussion of the results of a VS benchmark we performed to compare efficiency of the ligand-optimized receptors in comparison with the regular homology models. We observed that application of the ligand-optimized models significantly improved the VS performance both in terms of BEDROC (0.325 vs 0.182 for D1R and 0.383 vs 0.301 for D2R) as well as EF1% (20 vs 11 for D1R and 18 vs 10 for D2R). In contrast, no improvement was observed for the performance of a D2R model built on the D3R template, when compared with that derived from the structure of the previously published and more evolutionary distant ß2 adrenergic receptor. The comparison of results for receptors built according to various protocols and templates revealed that the most significant factor for the receptor performance was a proper selection of "tool ligand" used in induced fit docking procedure. Taken together, our results suggest that the described homology modeling procedure could be a viable tool for structure-based GPCR ligand design, even for the targets for which only a relatively distant structural template is available.

Correlation between in silico physicochemical characteristics of drugs and their mean residence time in human and dog.

The correlation between 52 physicochemical parameters and mean residence time (MRT) for 27 drugs used in human and dog were investigated. The physicochemical parameter values calculated provided a basis for deriving a series of arithmetic expressions, which were used to build a mathematical model describing the relationship between them and the MRT values. From the entire set of analyzed parameters, a subset of 14 was identified that contributed to the derivation of an arithmetic expression: Log(PSA - WPSA + ACID) x [XlogP - (LogKp - EAxLn(Caco2 + AMINE + SAF))] + (AMIDE + IP - FG) - Ln(MW + PISA) the value of which is highly correlated with the MRT value in dogs (P < .001) and allowed prediction of the MRT predicted (MRT(pred)). In humans, no correlation was found that allowed the calculation of MRT(pred). These results indicate that predicting the pharmacokinetics of any specific drug for humans based on pharmacokinetic data obtained in the dog should be undertaken with knowledge of the inherent limitations.

Structural model for the multisubunit Type IC restriction-modification DNA methyltransferase M.EcoR124I in complex with DNA.

Recent publication of crystal structures for the putative DNA-binding subunits (HsdS) of the functionally uncharacterized Type I restriction-modification (R-M) enzymes MjaXIP and MgeORF438 have provided a convenient structural template for analysis of the more extensively characterized members of this interesting family of multisubunit molecular motors. Here, we present a structural model of the Type IC M.EcoR124I DNA methyltransferase (MTase), comprising the HsdS subunit, two HsdM subunits, the cofactor AdoMet and the substrate DNA molecule. The structure was obtained by docking models of individual subunits generated by fold-recognition and comparative modelling, followed by optimization of inter-subunit contacts by energy minimization. The model of M.EcoR124I has allowed identification of a number of functionally important residues that appear to be involved in DNA-binding. In addition, we have mapped onto the model the location of several new mutations of the hsdS gene of M.EcoR124I that were produced by misincorporation mutagenesis within the central conserved region of hsdS, we have mapped all previously identified DNA-binding mutants of TRD2 and produced a detailed analysis of the location of surface-modifiable lysines. The model structure, together with location of the mutant residues, provides a better background on which to study protein-protein and protein-DNA interactions in Type I R-M systems.

MODOMICS: a database of RNA modification pathways.

MODOMICS is the first comprehensive database resource for systems biology of RNA modification. It integrates information about the chemical structure of modified nucleosides, their localization in RNA sequences, pathways of their biosynthesis and enzymes that carry out the respective reactions. MODOMICS also provides literature information, and links to other databases, including the available protein sequence and structure data. The current list of modifications and pathways is comprehensive, while the dataset of enzymes is limited to Escherichia coli and Saccharomyces cerevisiae and sequence alignments are presented only for tRNAs from these organisms. RNAs and enzymes from other organisms will be included in the near future. MODOMICS can be queried by the type of nucleoside (e.g. A, G, C, U, I, m1A, nm5s2U, etc.), type of RNA, position of a particular nucleoside, type of reaction (e.g. methylation, thiolation, deamination, etc.) and name or sequence of an enzyme of interest. Options for data presentation include graphs of pathways involving the query nucleoside, multiple sequence alignments of RNA sequences and tabular forms with enzyme and literature data. The contents of MODOMICS can be accessed through the World Wide Web at http://genesilico.pl/modomics/.

Novel inhibitors of the rRNA ErmC' methyltransferase to block resistance to macrolides, lincosamides, streptogramine B antibiotics.

In erythromycin-resistant bacteria, the N6 position of A2058 in 23S rRNA is mono- or dimethylated by Erm family methyltransferases. This modification results in cross-resistance to macrolides, lincosamides and streptogramin B. Most inhibitors of Erm methyltransferases developed up-to-date target the cofactor-binding pocket, resulting in a lack of selectivity whereas inhibitors that bind the substrate-binding pocket demonstrate low in vitro activity. In this study, a molecular docking approach followed by biochemical screening was applied to search for inhibitors targeting both cofactor- and substrate-binding pockets of ErmC' methyltransferase. Based on the results of the molecular docking-based virtual screening of the clean-leads subset of the ZINC database, 29 compounds were chosen for experimental verification. Among them inhibitor 28 (ZINC code 32747906), with an IC50 of 100 µM, decreased the minimal inhibitory concentration of erythromycin in the Escherichia coli strain overexpressing ErmC'. Docking analysis of 28 to the ErmC' structure and the competitive ligand binding assay revealed a non-competitive model of inhibition. Inhibitor 28 served as a template for similarity-based virtual screening, which resulted in the identification of two derivatives 3s (ZINC code 62022572) and 4s (ZINC code 49032257) with an IC50 of 116 µM and 110 µM, respectively. Our results provide a basis for the development of inhibitors against the Erm-family of enzymes.

Characterization of prmt7alpha and beta isozymes from Chinese hamster cells sensitive and resistant to topoisomerase II inhibitors.

By selection of genetic suppressor elements (GSEs) conferring resistance to topoisomerase II inhibitors in Chinese hamster cells (DC-3F), we identified a gene encoding two proteins of 78 and 82 kDa which belong to the protein arginine methyltransferase (PRMT) family. Down-regulation of these enzymes (named PRMT7alpha and beta), either induced by an antisense GSE or as observed in the 9-OH-ellipticine (9-OH-E) resistant mutant DC-3F/9-OH-E, was responsible for cell resistance to various DNA damaging agents. Alternative splicing alterations in the 5'-terminal region and changes of the polyadenylation site of PRMT7 mRNAs were observed in these resistant mutant cells. PRMT7alpha and beta are isoforms of a highly conserved protein containing two copies of a module common to all PRMTs, comprising a Rossmann-fold domain and a beta-barrel domain. The C-terminal repeat appears to be degenerate and catalytically inactive. PRMT7alpha and beta form homo- and hetero-dimers but differ by their sub-cellular localization and in vitro recognize different substrates. PRMT7beta was only observed in Chinese hamster cells while mouse 10T1/2 fibroblasts only contain PRMT7alpha. Surprisingly, in human cells the anti-PRMT7 antibody essentially recognized an approximately 37 kDa peptide, which is not formed during extraction, and a faint band at 78 kDa. Analysis of in vitro and in vivo methylation patterns in cell lines under- or over-expressing PRMT7alpha and beta detected a discrete number of proteins which methylation and/or expression are under the control of these enzymes.

Type II restriction endonuclease R.Eco29kI is a member of the GIY-YIG nuclease superfamily.

BACKGROUND: The majority of experimentally determined crystal structures of Type II restriction endonucleases (REases) exhibit a common PD-(D/E)XK fold. Crystal structures have been also determined for single representatives of two other folds: PLD (R.BfiI) and half-pipe (R.PabI), and bioinformatics analyses supported by mutagenesis suggested that some REases belong to the HNH fold. Our previous bioinformatic analysis suggested that REase R.Eco29kI shares sequence similarities with one more unrelated nuclease superfamily, GIY-YIG, however so far no experimental data were available to support this prediction. The determination of a crystal structure of the GIY-YIG domain of homing endonuclease I-TevI provided a template for modeling of R.Eco29kI and prompted us to validate the model experimentally. RESULTS: Using protein fold-recognition methods we generated a new alignment between R.Eco29kI and I-TevI, which suggested a reassignment of one of the putative catalytic residues. A theoretical model of R.Eco29kI was constructed to illustrate its predicted three-dimensional fold and organization of the active site, comprising amino acid residues Y49, Y76, R104, H108, E142, and N154. A series of mutants was constructed to generate amino acid substitutions of selected residues (Y49A, R104A, H108F, E142A and N154L) and the mutant proteins were examined for their ability to bind the DNA containing the Eco29kI site 5'-CCGCGG-3' and to catalyze the cleavage reaction. Experimental data reveal that residues Y49, R104, E142, H108, and N154 are important for the nuclease activity of R.Eco29kI, while H108 and N154 are also important for specific DNA binding by this enzyme. CONCLUSION: Substitutions of residues Y49, R104, H108, E142 and N154 predicted by the model to be a part of the active site lead to mutant proteins with strong defects in the REase activity. These results are in very good agreement with the structural model presented in this work and with our prediction that R.Eco29kI belongs to the GIY-YIG superfamily of nucleases. Our study provides the first experimental evidence for a Type IIP REase that does not belong to the PD-(D/E)XK or HNH superfamilies of nucleases, and is instead a member of the unrelated GIY-YIG superfamily.

Phylogenomic analysis of the GIY-YIG nuclease superfamily.

BACKGROUND: The GIY-YIG domain was initially identified in homing endonucleases and later in other selfish mobile genetic elements (including restriction enzymes and non-LTR retrotransposons) and in enzymes involved in DNA repair and recombination. However, to date no systematic search for novel members of the GIY-YIG superfamily or comparative analysis of these enzymes has been reported. RESULTS: We carried out database searches to identify all members of known GIY-YIG nuclease families. Multiple sequence alignments together with predicted secondary structures of identified families were represented as Hidden Markov Models (HMM) and compared by the HHsearch method to the uncharacterized protein families gathered in the COG, KOG, and PFAM databases. This analysis allowed for extending the GIY-YIG superfamily to include members of COG3680 and a number of proteins not classified in COGs and to predict that these proteins may function as nucleases, potentially involved in DNA recombination and/or repair. Finally, all old and new members of the GIY-YIG superfamily were compared and analyzed to infer the phylogenetic tree. CONCLUSION: An evolutionary classification of the GIY-YIG superfamily is presented for the very first time, along with the structural annotation of all (sub)families. It provides a comprehensive picture of sequence-structure-function relationships in this superfamily of nucleases, which will help to design experiments to study the mechanism of action of known members (especially the uncharacterized ones) and will facilitate the prediction of function for the newly discovered ones.

Sequence-structure-function studies of tRNA:m5C methyltransferase Trm4p and its relationship to DNA:m5C and RNA:m5U methyltransferases.

Three types of methyltransferases (MTases) generate 5-methylpyrimidine in nucleic acids, forming m5U in RNA, m5C in RNA and m5C in DNA. The DNA:m5C MTases have been extensively studied by crystallographic, biophysical, biochemical and computational methods. On the other hand, the sequence-structure-function relationships of RNA:m5C MTases remain obscure, as do the potential evolutionary relationships between the three types of 5-methylpyrimidine-generating enzymes. Sequence analyses and homology modeling of the yeast tRNA:m5C MTase Trm4p (also called Ncl1p) provided a structural and evolutionary platform for identification of catalytic residues and modeling of the architecture of the RNA:m5C MTase active site. The analysis led to the identification of two invariant residues that are important for Trm4p activity in addition to the conserved Cys residues in motif IV and motif VI that were previously found to be critical. The newly identified residues include a Lys residue in motif I and an Asp in motif IV. A conserved Gln found in motif X was found to be dispensable for MTase activity. Locations of essential residues in the model of Trm4p are in very good agreement with the X-ray structure of an RNA:m5C MTase homolog PH1374. Theoretical and experimental analyses revealed that RNA:m5C MTases share a number of features with either RNA:m5U MTases or DNA:m5C MTases, which suggested a tentative phylogenetic model of relationships between these three classes of 5-methylpyrimidine MTases. We infer that RNA:m5C MTases evolved from RNA:m5U MTases by acquiring an additional Cys residue in motif IV, which was adapted to function as the nucleophilic catalyst only later in DNA:m5C MTases, accompanied by loss of the original Cys from motif VI, transfer of a conserved carboxylate from motif IV to motif VI and sequence permutation.

Alanine-scanning mutagenesis of the predicted rRNA-binding domain of ErmC' redefines the substrate-binding site and suggests a model for protein-RNA interactions.

The Erm family of adenine-N(6) methyltransferases (MTases) is responsible for the development of resistance to macrolide-lincosamide-streptogramin B antibiotics through the methylation of 23S ribosomal RNA. Hence, these proteins are important potential drug targets. Despite the availability of the NMR and crystal structures of two members of the family (ErmAM and ErmC', respectively) and extensive studies on the RNA substrate, the substrate-binding site and the amino acids involved in RNA recognition by the Erm MTases remain unknown. It has been proposed that the small C-terminal domain functions as a target-binding module, but this prediction has not been tested experimentally. We have undertaken structure-based mutational analysis of 13 charged or polar residues located on the predicted rRNA-binding surface of ErmC' with the aim to identify the area of protein-RNA interactions. The results of in vivo and in vitro analyses of mutant protein suggest that the key RNA-binding residues are located not in the small domain, but in the large catalytic domain, facing the cleft between the two domains. Based on the mutagenesis data, a preliminary three-dimensional model of ErmC' complexed with the minimal substrate was constructed. The identification of the RNA-binding site of ErmC' may be useful for structure-based design of novel drugs that do not necessarily bind to the cofactor-binding site common to many S-adenosyl-L- methionine-dependent MTases, but specifically block the substrate-binding site of MTases from the Erm family.

The PD-(D/E)XK superfamily revisited: identification of new members among proteins involved in DNA metabolism and functional predictions for domains of (hitherto) unknown function.

BACKGROUND: The PD-(D/E)XK nuclease superfamily, initially identified in type II restriction endonucleases and later in many enzymes involved in DNA recombination and repair, is one of the most challenging targets for protein sequence analysis and structure prediction. Typically, the sequence similarity between these proteins is so low, that most of the relationships between known members of the PD-(D/E)XK superfamily were identified only after the corresponding structures were determined experimentally. Thus, it is tempting to speculate that among the uncharacterized protein families, there are potential nucleases that remain to be discovered, but their identification requires more sensitive tools than traditional PSI-BLAST searches. RESULTS: The low degree of amino acid conservation hampers the possibility of identification of new members of the PD-(D/E)XK superfamily based solely on sequence comparisons to known members. Therefore, we used a recently developed method HHsearch for sensitive detection of remote similarities between protein families represented as profile Hidden Markov Models enhanced by secondary structure. We carried out a comparison of known families of PD-(D/E)XK nucleases to the database comprising the COG and PFAM profiles corresponding to both functionally characterized as well as uncharacterized protein families to detect significant similarities. The initial candidates for new nucleases were subsequently verified by sequence-structure threading, comparative modeling, and identification of potential active site residues. CONCLUSION: In this article, we report identification of the PD-(D/E)XK nuclease domain in numerous proteins implicated in interactions with DNA but with unknown structure and mechanism of action (such as putative recombinase RmuC, DNA competence factor CoiA, a DNA-binding protein SfsA, a large human protein predicted to be a DNA repair enzyme, predicted archaeal transcription regulators, and the head completion protein of phage T4) and in proteins for which no function was assigned to date (such as YhcG, various phage proteins, novel candidates for restriction enzymes). Our results contributes to the reduction of "white spaces" on the sequence-structure-function map of the protein universe and will help to jump-start the experimental characterization of new nucleases, of which many may be of importance for the complete understanding of mechanisms that govern the evolution and stability of the genome.

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.

FRankenstein becomes a cyborg: the automatic recombination and realignment of fold recognition models in CASP6.

In the course of CASP6, we generated models for all targets using a new version of the "FRankenstein's monster approach." Previously (in CASP5) we were able to build many very accurate full-atom models by selection and recombination of well-folded fragments obtained from crude fold recognition (FR) results, followed by optimization of the sequence-structure fit and assessment of alternative alignments on the structural level. This procedure was however very arduous, as most of the steps required extensive visual and manual input from the human modeler. Now, we have automated the most tedious steps, such as superposition of alternative models, extraction of best-scoring fragments, and construction of a hybrid "monster" structure, as well as generation of alternative alignments in the regions that remain poorly scored in the refined hybrid model. We have also included the ROSETTA method to construct those parts of the target for which no reasonable structures were generated by FR methods (such as long insertions and terminal extensions). The analysis of successes and failures of the current version of the FRankenstein approach in modeling of CASP6 targets reveals that the considerably streamlined and automated method performs almost as well as the initial, mostly manual version, which suggests that it may be a useful tool for accurate protein structure prediction even in the hands of nonexperts.

Identification of a new family of putative PD-(D/E)XK nucleases with unusual phylogenomic distribution and a new type of the active site.

BACKGROUND: Prediction of structure and function for uncharacterized protein families by identification of evolutionary links to characterized families and known structures is one of the cornerstones of genomics. Theoretical assignment of three-dimensional folds and prediction of protein function even at a very general level can facilitate the experimental determination of the molecular mechanism of action and the role that members of a given protein family fulfill in the cell. Here, we predict the three-dimensional fold and study the phylogenomic distribution of members of a large family of uncharacterized proteins classified in the Clusters of Orthologous Groups database as COG4636. RESULTS: Using protein fold-recognition we found that members of COG4636 are remotely related to Holliday junction resolvases and other nucleases from the PD-(D/E)XK superfamily. Structure modeling and sequence analyses suggest that most members of COG4636 exhibit a new, unusual variant of the putative active site, in which the catalytic Lys residue migrated in the sequence, but retained similar spatial position with respect to other functionally important residues. Sequence analyses revealed that members of COG4636 and their homologs are found mainly in Cyanobacteria, but also in other bacterial phyla. They undergo horizontal transfer and extensive proliferation in the colonized genomes; for instance in Gloeobacter violaceus PCC 7421 they comprise over 2% of all protein-encoding genes. Thus, members of COG4636 appear to be a new type of selfish genetic elements, which may fulfill an important role in the genome dynamics of Cyanobacteria and other species they invaded. Our analyses provide a platform for experimental determination of the molecular and cellular function of members of this large protein family. CONCLUSION: After submission of this manuscript, a crystal structure of one of the COG4636 members was released in the Protein Data Bank (code 1wdj; Idaka, M., Wada, T., Murayama, K., Terada, T., Kuramitsu, S., Shirouzu, M., Yokoyama, S.: Crystal structure of Tt1808 from Thermus thermophilus Hb8, to be published). Our analysis of the Tt1808 structure reveals that we correctly predicted all functionally important features of the COG4636 family, including the membership in the PD-(D/E)xK superfamily of nucleases, the three-dimensional fold, the putative catalytic residues, and the unusual configuration of the active site.

Mutational analysis defines the roles of conserved amino acid residues in the predicted catalytic pocket of the rRNA:m6A methyltransferase ErmC'.

Methyltransferases (MTases) from the Erm family catalyze S-adenosyl-L-methionine-dependent modification of a specific adenine residue in bacterial 23S rRNA, thereby conferring resistance to clinically important macrolide, lincosamide and streptogramin B antibiotics. Despite the available structural data and functional analyses on the level of the RNA substrate, still very little is known about the mechanism of rRNA:adenine-N(6) methylation. Only predictions regarding various aspects of this reaction have been made based on the analysis of the crystal structures of methyltransferase ErmC' (without the RNA) and their comparison with the crystallographic and biochemical data for better studied DNA:m(6)A MTases. To validate the structure-based predictions of presumably essential residues in the catalytic pocket of ErmC', we carried out the site-directed mutagenesis and studied the function of the mutants in vitro and in vivo. Our results indicate that the active site of rRNA:m(6)A MTases is much more tolerant to amino acid substitutions than the active site of DNA:m(6)A MTases. Only the Y104 residue implicated in stabilization of the target base was found to be indispensable. Remarkably, the N101 residue from the "catalytic" motif IV and two conserved residues that form the floor (F163) and one of the walls (N11) of the base-binding site are not essential for catalysis in ErmC'. This somewhat surprising result is discussed in the light of the available structural data and in the phylogenetic context of the Erm family.

Characterization of the cofactor-binding site in the SPOUT-fold methyltransferases by computational docking of S-adenosylmethionine to three crystal structures.

BACKGROUND: There are several evolutionarily unrelated and structurally dissimilar superfamilies of S-adenosylmethionine (AdoMet)-dependent methyltransferases (MTases). A new superfamily (SPOUT) has been recently characterized on a sequence level and three structures of its members (1gz0, 1ipa, and 1k3r) have been solved. However, none of these structures include the cofactor or the substrate. Due to the strong evolutionary divergence and the paucity of experimental information, no confident predictions of protein-ligand and protein-substrate interactions could be made, which hampered the study of sequence-structure-function relationships in the SPOUT superfamily. RESULTS: We used the computational docking program AutoDock to identify the AdoMet-binding site on the surface of three MTase structures. We analyzed the sequence divergence in two distinct lineages of the SPOUT superfamily in the context of surface features and preferred cofactor binding mode to propose specific function for the conserved residues. CONCLUSION: Our docking analysis has confidently predicted the common AdoMet-binding site in three remotely related proteins structures. In the vicinity of the cofactor-binding site, subfamily-conserved grooves were identified on the protein surface, suggesting location of the target-binding/catalytic site. Functionally important residues were inferred and a general reaction mechanism, involving conformational change of a glycine-rich loop, was proposed.

A "FRankenstein's monster" approach to comparative modeling: merging the finest fragments of Fold-Recognition models and iterative model refinement aided by 3D structure evaluation.

We applied a new multi-step protocol to predict the structures of all targets during CASP5, regardless of their potential category. 1) We used diverse fold-recognition (FR) methods to generate initial target-template alignments, which were converted into preliminary full-atom models by comparative modeling. All preliminary models were evaluated (scored) by VERIFY3D to identify well- and poorly-folded fragments. 2) Preliminary models with similar 3D folds were superimposed, poorly-scoring regions were deleted and the "average model" structure was created by merging the remaining segments. All template structures reported by FR were superimposed and a composite multiple-structure template was created from the most conserved fragments. 3). The average model was superimposed onto the composite template and the structure-based target-template alignment was inferred. This alignment was used to build a new (intermediate) comparative model of the target, again scored with VERIFY3D. 4) For all poorly scoring regions series of alternative alignments were generated by progressively shifting the "unfit" sequence fragment in either direction. Here, we considered additional information, such as secondary structure, placement of insertions and deletions in loops, conservation of putative catalytic residues, and the necessity to obtain a compact, well-folded structure. For all alternative alignments, new models were built and evaluated. 5) All models were superimposed and the "FRankenstein's monster" (FR, fold recognition) model was built from best-scoring segments. The final model was obtained after limited energy minimization to remove steric clashes between sidechains from different fragments. The novelty of this approach is in the focus on "vertical" recombination of structure fragments, typical for the ab initio field, rather than "horizontal" sequence alignment typical for comparative modeling. We tested the usefulness of the "FRankenstein" approach for non-expert predictors: only the leader of our team had considerable experience in protein modeling - he registered as a separate group (020) and submitted models built only by himself. At the onset of CASP5, the other five members of the team (students) had very little or no experience with modeling. They followed the same protocol in a deliberately naïve way. In the fourth step they used solely the VERIFY3D criterion to compare their models and the leader's model (the latter regarded only as one of the many alternatives) and generated the hybrid or selected only one model for submission (group 517). In order to compare our protocol with the traditional "one target-one template-one alignment" approach, we submitted (as a separate group 242) models selected from those automatically generated by all CAFASP servers (i.e. obtained without any human intervention). Here, we compare the results obtained by the three "groups", describe successes and failures of the "FRankenstein" approach and discuss future developments of comparative modeling. The automatic version of our multi-step protocol is being developed as a meta-server; the prototype is freely available at http://genesilico.pl/meta/.

Molecular phylogenetics of the RrmJ/fibrillarin superfamily of ribose 2'-O-methyltransferases.

Recent analyses identified a putative catalytic tetrad K-D-K-E common to several families of site-specific methyltransferases (MTases) that modify 2'-hydroxyl groups of ribose in mRNA, rRNA and tRNA (designated the RrmJ class after one of the structurally characterized members; 1eiz in Protein Data Bank) [Genome Biol. 2(9) (2001) 38]. Subsequently, three residues of the tetrad (K-D-K) were shown to be essential for catalysis in RrmJ [J. Biol. Chem. 277 (2002) 41978]. Here, we report identification of a similar conserved tetrad (K-D-K-H) in the family of snoRNA-guided ribose 2'-O-MTases related to fibrillarin (represented by the Mj0697 protein structure; 1fbn in PDB). The corresponding functional groups of putative catalytic tetrads of RrmJ and Mj0697 may be superimposed in space. However, one of the invariant residues (K(164) in RrmJ and K(179) in Mj0697) is observed in two distinct locations in the primary sequence, suggesting an interesting case of 'migration' of the conserved side chain within the framework of the active site. RrmJ and Mj0697 sequences were used as starting points to carry out comprehensive sequence database searches, resulting in identification of a similar conserved tetrad (and hence, prediction of a ribose 2'-O-specificity) in several families of putative MTases, including TlyA hemolysins, novel proteins from Trypanosoma, and large multidomain proteins from Flaviviriruses, Nidoviruses, and Alphaviruses. The results of our analysis of phylogenetic relationships in the RrmJ/fibrillarin superfamily provide insight into the evolution of site-specific and snoRNA-guided ribose 2'-O-MTases from a common ancestor.

Qualitative structure residue relationship analysis in the determination of the maximum residue limit of veterinary drugs.

Aim of the present study was an attempt to find a correlation between physicochemical structure of veterinary drugs and the maximum residue limit (MRL) for muscle tissue of food producing animals. Direct correlation and analysis in quintile groups for 52 physicochemical parameters were performed. An internal validation using leave-one-out cross-validation was performed. In the quintile groups, there were 11 arithmetic expressions created for the limited group of individual parameters (13 from 52 analyzed), which showed a significant linear or quadratic correlation between the number of quintile group and the mean value of MRL within the quintile. The results obtained suggest that there is no direct correlation between individual physicochemical parameters and MRL value in muscle tissue; however, such correlation can be determined for arithmetic expressions created on the basis of several physicochemical parameters, using quintile group analysis.