Comparative modeling of CASP4 target proteins: combining results of sequence search with three-dimensional structure assessment.

Comparative modeling aims at constructing molecular models for proteins of unknown structure, by using known structures of related proteins as templates. To test the comparative modeling approach reported here, predictions for 13 target proteins were submitted during the fourth round of "blind" protein structure prediction experiment (CASP4; http://PredictionCenter.llnl.gov/casp4). Sequence identity between these target proteins and the closest known structures ranged from 13 to 58%, indicating a broad spectrum of prediction difficulty. Although this broad difficulty range required addressing a variety of issues, the most important proved to be sequence-structure alignment for distant homology targets. The alignment step was based on structure-based evaluation of alignment variants produced mainly with PSI-BLAST intermediate sequence search procedure (PSI-BLAST-ISS). Although a fraction of correctly aligned residues in resulting models was markedly better than the average in all cases, for distant homology targets it was still considerably below the estimated achievable level. Results with CASP4 targets show that, along with the correctness of sequence-structure alignments, effective use of multiple template structures may significantly increase accuracy of the model structure. Improvement in this area should also result in more accurate loop modeling and side-chain prediction.

Comparison of performance in successive CASP experiments.

As the number of completed CASP (Critical Assessment of Protein Structure Prediction) experiments grows, so does the need for stable, standard methods for comparing performance in successive experiments. It is critical to develop methods for determining the areas in which there is progress and in which areas are static. We have added an analysis of the CASP4 results to that previously published for CASPs 1, 2, and 3. We again use a unified difficulty scale to permit comparison of performance as a function of target difficulty in the different CASPs. The scale is used to compare performance in aligning target sequences to a structural template. There was a clear improvement in alignment quality between CASP1 (1994) and CASP2 (1996). No change is apparent between CASP2 and CASP3 (1998). There is a small barely detectable improvement between CASP3 and the latest experiment (CASP4, 2000). Alignment remains the major source of error in all models based on less than about 30% sequence identity. Comparison of performance in the new fold modeling regime is complicated by issues in devising an objective target difficulty scale. We have found limited numerical support for significant progress between CASP3 and CASP4 in this area. More subjectively, most observers are convinced that there has been substantial progress. Progress is dominated by a single group.

Structure-based sequence alignment for the beta-trefoil subdomain of the clostridial neurotoxin family provides residue level information about the putative ganglioside binding site.

Clostridial neurotoxins embrace a family of extremely potent toxins comprised of tetanus toxin (TeNT) and seven different serotypes of botulinum toxin (BoNT/A-G). The beta-trefoil subdomain of the C-terminal part of the heavy chain (H(C)), responsible for ganglioside binding, is the most divergent region in clostridial neurotoxins with sequence identity as low as 15%. We re-examined the alignment between family sequences within this subdomain, since in this region all alignments published to date show obvious inconsistencies with the beta-trefoil fold. The final alignment was obtained by considering the general constraints imposed by this fold, and homology modeling studies based on the TeNT structure. Recently solved structures of BoNT/A confirm the validity of this structure-based approach. Taking into account biochemical data and crystal structures of TeNT and BoNT/A, we also re-examined the location of the putative ganglioside binding site and, using the new alignment, characterized this site in other BoNT serotypes.

Structure-based predictions of Rad1, Rad9, Hus1 and Rad17 participation in sliding clamp and clamp-loading complexes.

The repair of damaged DNA is coupled to the completion of DNA replication by several cell cycle checkpoint proteins, including, for example, in fission yeast Rad1(Sp), Hus1(Sp), Rad9(Sp) and Rad17(Sp). We have found that these four proteins are conserved with protein sequences throughout eukaryotic evolution. Using computational techniques, including fold recognition, comparative modeling and generalized sequence profiles, we have made high confidence structure predictions for the each of the Rad1, Hus1 and Rad9 protein families (Rad17(Sc), Mec3(Sc) and Ddc1(Sc) in budding yeast, respectively). Each of these families was found to share a common protein fold with that of PCNA, the sliding clamp protein that tethers DNA polymerase to its template. We used previously reported genetic and biochemical data for these proteins from yeast and human cells to predict a heterotrimeric PCNA-like ring structure for the functional Rad1/Rad9/Hus1 complex and to determine their exact order within it. In addition, for each individual protein family, contact regions with neighbors within the PCNA-like ring were identified. Based on a molecular model for Rad17(Sp), we concluded that members of this family, similar to the subunits of the RFC clamp-loading complex, are capable of coupling ATP binding with conformational changes required to load a sliding clamp onto DNA. This model substantiates previous findings regarding the behavior of Rad17 family proteins upon DNA damage and within the RFC complex of clamp-loading proteins.

Processing and analysis of CASP3 protein structure predictions.

Livermore Prediction Center provides basic infrastructure for the CASP (Critical Assessment of Structure Prediction) experiments, including prediction processing and verification servers, a system of prediction evaluation tools, and interactive numerical and graphical displays. Here we outline the essentials of our approach, with discussion of the superposition procedures, definitions of basic measures, and descriptions of new methods developed to analyze predictions. Our primary focus is on the evaluation of three-dimensional models and secondary structure predictions. To put the results of the three prediction experiments held to date on the same footing, the latest CASP3 evaluation criteria were retrospectively applied to both CASP1 and CASP2 predictions. Finally, we give an overview of our website (http:/(/)PredictionCenter.llnl.gov), which makes the target structures, predictions, and the evaluation system accessible to the community.

Addressing the issue of sequence-to-structure alignments in comparative modeling of CASP3 target proteins.

During a blind protein structure prediction experiment (the third round of the Critical Assessment of Techniques for Protein Structure Prediction; URL http://PredictionCenter.llnl.gov/casp3/) , four target proteins, T0047, T0048, T0055, and T0070, were modeled by comparison. These proteins display 62%, 29%, 24%, and 19% sequence identity, respectively, to the structurally homologous proteins most similar in sequence. The issue of sequence-to-structure alignment in cases of low sequence homology was the main emphasis. Selection of alignments was made by constructing and evaluating three-dimensional models based on series of samples produced mainly by automatic multiple sequence alignments. Sequence-to-structure alignments were correct in all but two regions, in which significant changes in target structures compared with related proteins were the source of errors. Template choice is an important determinant of model quality, and a correct selection was made of a lower homology template for modeling of T0070; however, in the case of T0055, a template with 8% greater sequence homology proved deceptive. Loops and some ungapped template regions were assigned conformations taken from other proteins. Using fragments from homologous structures led to improvement over template backbone more often than cases in which nonhomologous structures were the source. The results also indicate that side-chain prediction accuracy depends not only on sequence similarity but also on accuracy of the backbone.

Some measures of comparative performance in the three CASPs.

Performance in the three Critical Assessment of protein Structure Prediction (CASP) experiments has been compared in the areas of alignment accuracy for models based on homology and three-dimensional accuracy for models produced by using ab initio prediction methods. The homologous models span the comparative modeling and fold-recognition regimes. Each CASP target is assigned a relative difficulty based on the extent of sequence identity and the degree of structural overlap with the best available template. There is a clear improvement in alignment accuracy between CASP1 and CASPs 2 and 3 over much of the difficulty scale but no apparent improvement between CASP2 and CASP3. Encouragingly, the best ab initio models of small targets are clearly more accurate in CASP3 than in CASPs 1 and 2.

A modified definition of Sov, a segment-based measure for protein secondary structure prediction assessment.

We present a measure for the evaluation of secondary structure prediction methods that is based on secondary structure segments rather than individual residues. The algorithm is an extension of the segment overlap measure Sov, originally defined by Rost et al. (J Mol Biol 1994;235:13-26). The new definition of Sov corrects the normalization procedure and improves Sov's ability to discriminate between similar and dissimilar segment distributions. The method has been comprehensively tested during the second Critical Assessment of Techniques for Protein Structure Prediction (CASP2). Here, we describe the underlying concepts, modifications to the original definition, and their significance.

Criteria for evaluating protein structures derived from comparative modeling.

Following the first experiment for the Critical Assessment of methods for protein Structure Prediction (CASP1), numerical criteria were devised to analyze the performance of prediction methods. We report here the criteria for comparative modeling, and how effective they were in CASP2. These criteria are intended to evolve into a set of numerical measures that provide a comprehensive assessment of the quality of a structure produced by comparative modeling, and provide a means of investigating which modeling methods are most effective, so as to establish where future effort may be most productively applied.

Numerical criteria for the evaluation of ab initio predictions of protein structure.

As part of the CASP2 protein structure prediction experiment, a set of numerical criteria were defined for the evaluation of "ab initio" predictions. The evaluation package comprises a series of electronic submission formats, a submission validator, evaluation software, and a series of scripts to summarize the results for the CASP2 meeting and for presentation via the World Wide Web (WWW). The evaluation package is accessible for use on new predictions via WWW so that results can be compared to those submitted to CASP2. With further input from the community, the evaluation criteria are expected to evolve into a comprehensive set of measures capturing the overall quality of a prediction as well as critical detail essential for further development of prediction methods. We discuss present measures, limitations of the current criteria, and possible improvements.

Different enzymes with similar structures involved in Mg(2+)-mediated polynucleotidyl transfer.

Comparison of X-ray structures of restriction endonucleases and polynucleotidyl transferase superfamily enzymes reveals a structural resemblance.

Five-stranded beta-sheet sandwiched with two alpha-helices: a structural link between restriction endonucleases EcoRI and EcoRV.

Examination of crystal structures of restriction endonucleases EcoRI and EcoRV complexes with their cognate DNA revealed a common structural element, which forms the core of both proteins. This element consists of a five-stranded beta-sheet and two alpha-helices packed against it and could be described as alpha-beta sandwich in which helices and beta-strands lie in two stacked layers. While the spatial structure of this alpha-beta sandwich is conserved in both enzymes, there are not detectable similarities between amino acid sequences except of a few residues involved in active site formation. Probably, other restriction endonucleases which have similar organization of the active site might possess similar structural element regardless of DNA sequence recognized and recognition elements in the enzyme used.

Codon-anticodon pairing. A model for interacting codon-anticodon duplexes located at the ribosomal A- and P-sites.

The interaction between two codon-anticodon duplexes of the ribosomal A- and P-site-bound tRNAs is the key feature of the proposed model. This interaction prohibits non-canonical base pairing at the first and second positions of the codon and controls base pairing at the third position (wobbling rules ensuing from the model are in good accord with those generated from experiments). The model is capable of predicting codon context effects. It follows from the model that modifications of the first anticodon residue of the P-site tRNA can affect the stability of the A-site duplex, and that the translation of a DNA single chain analogue of mRNA should be accompanied by non-canonical base pairing at all three positions of the codon. These predictions of the model can be subjected to experimental tests.

How are tRNAs and mRNA arranged in the ribosome? An attempt to correlate the stereochemistry of the tRNA-mRNA interaction with constraints imposed by the ribosomal topography.

Two tRNA molecules at the ribosomal A- and P-sites, with a relatively small angle between the planes of the L-shaped molecules, can be arranged in two mutually exclusive orientations. In one (the 'R'-configuration), the T-loop of the A-site tRNA faces the D-loop of the P-site tRNA, whereas in the other (the 'S'-configuration) the D-loop of the A-site tRNA faces the T-loop of the P-site tRNA. A number of stereochemical arguments, based on the crystal structure of 'free' tRNA, favour the R-configuration. In the ribosome, the CCA-ends of the tRNA molecules are 'fixed' at the base of the central protuberance (the peptidyl transferase centre) of the 50S subunit, and the anticodon loops lie in the neck region (the decoding site) of the 30S subunit. The translocation step is essentially a rotational movement of the tRNA from the A- to the P-site, and there is convincing evidence that the A-site must be located nearest to the L7/L12 protuberance of the 50S subunit. The mRNA in the two codon-anticodon duplexes lies on the 'inside' of the 'elbows' of the tRNA molecules (in both the S-type and R-type configurations), and runs up between the two molecules from the A- to the P-site in the 3' to 5'-direction. These considerations have the consequence that in the S-configuration the mRNA in the codon-anticodon duplexes is directed towards the 50S subunit, whereas in the R-configuration it is directed towards the 30S subunit. The results of site-directed cross-linking experiments, in particular cross-links to mRNA at positions within or very close to the codons interacting with A- or P-site tRNA, favour the latter situation. This conclusion is in direct contradiction to other current models for the arrangement of mRNA and tRNA on the ribosome.

The path of a protein chain can be approximated by the conformation dictated by interpeptide ionic bridges.

A stereochemical simulation of the formation of ionic bridges between adjacent peptide groups along the polypeptide chain has been made. Such ionic bridges constrain the amino-acid residues into eight conformations. It is shown that the path of any protein-chain fragment 10-15 residues long can be approximated well by these conformations. This suggests that the conformations dictated by the ionic bridges can be used as blocks in the formation of the spatial protein structure.