SiPep: a system for the prediction of tissue-specific minor histocompatibility antigens.

Approximately 50 years ago it was found that inbred strains of mice were able to reject tumours and skin grafts from major histocompatibility complex (MHC) identical donors. They proposed that additional transplantation antigens must exist outside the MHC. These were described as minor histocompatibility antigens (mHAgs). Since then, related studies in humans have identified 16 human mHAgs. The aim of this work is to increase the number of known mHAgs by prediction of candidate minor histocompatibility loci by identifying coding single nucleotide polymorphisms (SNPs) where the amino acid variation lies within an MHC-binding peptide and alters the ability of that peptide to bind. We have developed an algorithm called SiPep which uses peptide sequences derived from the flanking regions of known non-synonymous SNPs, various MHC-binding and proteolytic cleavage evaluation methods and protein expression data to predict mHAgs. We have processed 45094 SNPs using the SiPep algorithm and have stored the results in a database called SNPBinder. The facilities to process submitted proteins through the SiPep algorithm as well as the SNPBinder database are available to the public. A set of peptides that are predicted as possible mHAgs by the SiPep algorithm have been tested using refolding assays and gel filtration and the results are presented in this paper. The SiPep tools and SNPBinder database are available free of charge via the internet. An HTML interface providing search facilities can be found at the following address: http://www.sipep.org/.

The collation of forensic DNA case data into a multi-dimensional intelligence database.

The primary aim of any DNA Database is to link individuals to unsolved offenses and unsolved offenses to each other via DNA profiling. This aim has been successfully realised during the operation of the New Zealand (NZ) DNA Databank over the past five years. The DNA Intelligence Project (DIP), a collaborative project involving NZ forensic and law enforcement agencies, interrogated the forensic case data held on the NZ DNA databank and collated it into a functional intelligence database. This database has been used to identify significant trends which direct Police and forensic personnel towards the most appropriate use of DNA technology. Intelligence is being provided in areas such as the level of usage of DNA techniques in criminal investigation, the relative success of crime scene samples and the geographical distribution of crimes. The DIP has broadened the dimensions of the information offered through the NZ DNA Databank and has furthered the understanding and investigative capability of both Police and forensic scientists. The outcomes of this research fit soundly with the current policies of 'intelligence led policing', which are being adopted by Police jurisdictions locally and overseas.

The Bioinformatics Template Library--generic components for biocomputing.

MOTIVATION: The efficiency of bioinformatics programmers can be greatly increased through the provision of ready-made software components that can be rapidly combined, with additional bespoke components where necessary, to create finished programs. The new standard for C++ includes an efficient and easy to use library of generic algorithms and data-structures, designed to facilitate low-level component programming. The extension of this library to include functionality that is specifically useful in compute-intensive tasks in bioinformatics and molecular modelling could provide an effective standard for the design of reusable software components within the biocomputing community. RESULTS: A novel application of generic programming techniques in the form of a library of C++ components called the Bioinformatics Template Library (BTL) is presented. This library will facilitate the rapid development of efficient programs by providing efficient code for many algorithms and data-structures that are commonly used in biocomputing, in a generic form that allows them to be flexibly combined with application specific object-oriented class libraries. AVAILABILITY: The BTL is available free of charge from our web site http://www.cryst.bbk.ac.uk/~classlib/ and the EMBL file server http://www.embl-ebi.ac.uk/FTP/index.html

Opening of the active site of Clostridium perfringens alpha-toxin may be triggered by membrane binding.

On the basis of amino acid sequence homologies with other phospholipases C, the alpha-toxin of Clostridium perfringens was predicted to be a two-domain protein. Using truncated forms of alpha-toxin the phospholipase C active site was shown to be located in the amino-terminal domain. Crystallographic studies have confirmed this organisation and have also revealed that the carboxy-terminal domain is structurally similar to the phospholipid-binding domains in eukaryotic proteins. This information has been used to devise a model predicting how alpha-toxin interacts with membranes via calcium-mediated recognition of phospholipid head groups and the interaction of hydrophobic amino acids with the phospholipid tail group. The binding of alpha-toxin to membranes appears to result in the opening of the active site allowing hydrolysis of membrane phospholipids.

Rfree and the rfree ratio. II. Calculation Of the expected values and variances of cross-validation statistics in macromolecular least-squares refinement.

The free R factor is used routinely as a cross-validation tool in macromolecular crystallography. However, without any means of deriving quantitative estimates of its expected value and variance, its application has been rather subjective and its usefulness therefore somewhat limited. In the first part of this series, estimates of the expected value of the ratio of the free R factor to the standard R factor at the convergence of the structure refinement were given. Here, estimates of the variance of this ratio are given and are compared with the observed deviations from the expected values for a selection of refined structures. It is discussed how errors in the functional form of the structure-factor model as well as other types of errors might influence this ratio.

Identification of residues in the carboxy-terminal domain of Clostridium perfringens alpha-toxin (phospholipase C) which are required for its biological activities.

A panel of random mutants within the DNA encoding the carboxy-terminal domain of Clostridium perfringens alpha-toxin was constructed. Three mutants were identified which encoded alpha-toxin variants (Lys330Glu, Asp305Gly, and Asp293Ser) with reduced hemolytic activity. These variants also had diminished phospholipase C activity toward aggregated egg yolk phospholipid and reduced cytotoxic and myotoxic activities. Asp305Gly showed a significantly increased enzymatic activity toward the monodisperse substrate rhoNPPC, whereas Asp293Ser displayed a reduced activity toward this phospholipid analogue. In addition, Asp293Ser showed an increased dependence on calcium for enzymatic activity toward aggregated phospholipid and appeared calcium-depleted in PAGE band-shift assays. In contrast, neither Lys330Glu nor Asp305Gly showed altered dependence on calcium for enzymatic activity toward aggregated phospholipid. Asp305 is located in the interface between the amino- and carboxy-terminal domains, whereas Asp293 and Lys330 are surface exposed residues which may play a role in the recognition of membrane phospholipids.

Rfree and the rfree ratio. I. Derivation of expected values of cross-validation residuals used in macromolecular least-squares refinement.

The last five years have seen a large increase in the use of cross validation in the refinement of macromolecular structures using X-ray data. In this technique a test set of reflections is set aside from the working set and the progress of the refinement is monitored by the calculation of a free R factor which is based only on the excluded reflections. This paper gives estimates for the ratio of the free R factor to the R factor calculated from the working set for both unrestrained and restrained refinement. It is assumed that both the X-ray and restraint observations have been weighted correctly and that there is no correlation of errors between the test and working sets. It is also shown that the least-squares weights that minimize the variances of the refined parameters, also approximately minimize the free R factor. The estimated free R-factor ratios are compared with those reported for structures in the Protein Data Bank.

Error estimates of protein structure coordinates and deviations from standard geometry by full-matrix refinement of gammaB- and betaB2-crystallin.

Faster workstations with larger memories are making error estimation from full-matrix least-squares refinement a more practicable technique in protein crystallography. Using minimum variance weighting, estimated standard deviations of atomic positions have been calculated for two eye lens proteins from the inverse of a least-squares normal matrix which was full with respect to the coordinate parameters. gammaB-crystallin, refined at 1.49 A yielded average errors in atomic positions which ranged from 0.05 A for main-chain atoms to 0.27 A for unrestrained water molecules. The second structure used in this work was that of betaB2-crystallin refined at 2.1 A resolution where the corresponding average errors were 0.08 and 0.35 A, respectively. The relative errors in atomic positions are dependent on the number and kinds of restraints used in the refinements. It is also shown that minimum variance weighting leads to mean-square deviations from target geometry in the refined structures which are smaller than the variances used in the distance weighting.

Structure of the key toxin in gas gangrene.

Clostridium perfringens alpha-toxin is the key virulence determinant in gas gangrene and has also been implicated in the pathogenesis of sudden death syndrome in young animals. The toxin is a 370-residue, zinc metalloenzyme that has phospholipase C activity, and can bind to membranes in the presence of calcium. The crystal structure of the enzyme reveals a two-domain protein. The N-terminal domain shows an anticipated structural similarity to Bacillus cereus phosphatidylcholine-specific phospholipase C (PC-PLC). The C-terminal domain shows a strong structural analogy to eukaryotic calcium-binding C2 domains. We believe this is the first example of such a domain in prokaryotes. This type of domain has been found to act as a phospholipid and/or calcium-binding domain in intracellular second messenger proteins and, interestingly, these pathways are perturbed in cells treated with alpha-toxin. Finally, a possible mechanism for alpha-toxin attack on membrane-packed phospholipid is described, which rationalizes its toxicity when compared to other, non-haemolytic, but homologous phospholipases C.

Crystallization and preliminary X-ray diffraction studies of alpha-toxin from two different strains (NCTC8237 and CER89L43) of Clostridium perfringens.

The alpha-toxin of Clostridium perfringens is the major virulence determinant for gas gangrene in man. The gene encoding the alpha-toxin has been cloned into E. coli from two strains of the bacterium (NCTC8237 and CER89L43) and subsequently purified to homogeneity. The two strains of alpha-toxin differ by five amino acids, resulting in the toxin from NCTC8237 being sensitive to chymotrypsin digestion while that from CER89L43 is resistant. The alpha-toxin from each of these strains has been crystallized in two different forms by the hanging-drop vapour-diffusion method at 293 K. CER89L43 form I crystals belong to space group R32 and have two molecules in the crystallographic asymmetric unit and a unit cell with a = b = 151.4, c = 195.5 A, alpha = beta = 90, gamma = 120 degrees. The crystals diffracted to dmin = 1.90 A. The characteristics of the NCTC8237 form I crystals have already been reported. The form II crystals from both strains belong to space group C2221 with one molecule in the crystallographic asymmetric unit and, for strain CER89L43, have cell dimensions a = 61.05, b = 177.50, c = 79.05 A, alpha = beta = gamma = 90 degrees, while for strain NCTC8237 the cell dimensions are a = 60.50, b = 175.70, c = 80.20 A, alpha = beta = gamma = 90 degrees. The crystals diffracted to maximum resolutions of 1.85 and 2.1 A for the CER89L43 and the NCTC8237 strains, respectively.

Close packing of an oligomeric eye lens beta-crystallin induces loss of symmetry and ordering of sequence extensions.

beta-Crystallins are oligomeric eye lens proteins that are related to monomeric gamma-crystallins. The main sequence difference between the two families is the presence of sequence extensions in the beta-crystallins. A major question concerns the role that these extensions play in mediating interactions at the high protein concentrations found in the lens. The predominant beta-crystallin polypeptide, beta B2, can be crystallized in two different space groups, I222 and C222. The I222 crystal structure revealed that the protein packed as a tetramer with perfect 222 symmetry but that the extensions were disordered. The X-ray structure of the C222 lattice of beta B2 has now been refined at 3.3 A, the structure analysed and compared with the I222 lattice. The protein is also a tetramer with 222 symmetry in the C222 lattice but differs in that parts of the N-terminal extensions have been visualized. In the asymmetric unit of the C222 lattice there are four subunits, each comprising a single polypeptide chain, in which certain flexible loops in the N-terminal domains and the N-terminal extensions have various conformations. The tetramers in the C222 lattice are more tightly packed than in the I222 form. Analysis of the tetramer contacts shows that the sites of interaction break the 222 symmetry of the tetramers. The N-terminal extensions play a major role in directing interactions between tetramers. One of the N-terminal extensions interacts with a hydrophobic patch on the N-terminal domain of another tetramer. These crystallographic observations obtained over a physiological concentration range indicate how, in beta-crystallin oligomers, the N-terminal extensions of beta B2 can switch from interacting with water to interacting with protein depending on their relative concentrations. This could be useful in maintaining a gradient of refractive index.

An algorithm for automatically generating protein topology cartoons.

An algorithm is described for automatically generating protein topology cartoons. This algorithm optimally places circles and triangles depicting alpha-helices and beta-strands respectively giving a pictorial topological summary of any protein structure. beta-Sheets, sandwiches and barrels are automatically identified and represented using special templates. The output from this algorithm may be controlled by adjustment of variable weights during the optimization step giving a preferred result. The rules for generating protein toplogy cartoons, including consideration of the handedness of local structure motifs, are discussed. The design of this algorithm is completely general and is easily adapted to include further rules that dictate the generation of the cartoons.

Comparison of conformational characteristics in structurally similar protein pairs.

Although it is known that three-dimensional structure is well conserved during the evolutionary development of proteins, there have been few studies that consider other parameters apart from divergence of the main-chain coordinates. In this study, we align the structures of 90 pairs of homologous proteins having sequence identities ranging from 5 to 100%. Their structures are compared as a function of sequence identity, including not only consideration of C alpha coordinates but also accessibility, Ooi numbers, secondary structure, and side-chain angles. We discuss how these properties change as the sequences become less similar. This will be of practical use in homology modeling, especially for modeling very distantly related or analogous proteins. We also consider how the average size and number of insertions and deletions vary as sequences diverge. This study presents further quantitative evidence that structure is remarkably well conserved in detail, as well as at the topological level, even when the sequences do not show similarity that is significant statistically.

Main-chain bond lengths and bond angles in protein structures.

The main-chain bond lengths and bond angles of protein structures are analysed as a function of resolution. Neither the means nor standard deviations of these parameters show any correlation with resolution over the resolution range investigated. This is as might be expected as bond lengths and bond angles are likely to be heavily influenced by the geometrical restraints applied during structure refinement. The size of this influence is then investigated by performing an analysis of variance on the mean values across the five most commonly used refinement methods. The differences in means are found to be highly statistically significant, suggesting that the different target values used by the different methods leave their imprint on the structures they refine. This has implications concerning the actual target values used during refinement and stresses the importance of the values being not only accurate but also consistent from one refinement method to another.

Structure of the bovine eye lens protein gammaB(gammaII)-crystallin at 1.47 A.

The molecular structure of calf gammaB-crystallin (previously called gammaII), a lens-specific protein, has been refined to a crystallographic R factor of 18.1% for all reflection data, between 8.0 and 1.47 A, 25 959 hkl measured at 293 (1) K. 230 water molecules have been defined by difference Fourier techniques and included in a restrained least-squares refinement. Difference Fourier maps clearly indicated the presence of multiple sites for the sulfur atoms of Cys 18 and Cys 22 which were therefore given coupled second-site occupancies during the refinement. The sulfur atom in the major position of Cys 22 is in the reduced state. Either of the Cys 18 sites can form a high-energy disulfide bridge with the minor position of Cys 22. The position of the carboxy terminus and many other surface side chains have been further defined including the RGD signal peptide. The hydration of the backbone and the interdomain region has been analysed. 27 water molecules make extensive contacts to a single protein molecule and thus contribute to its stability.

Newly observed binding mode in pancreatic ribonuclease.

Dinucleotides containing guanine, when soaked into crystals of bovine pancreatic ribonuclease, have been found to bind in an unexpected manner, quite unlike interpretations of earlier X-ray diffraction studies. This finding has prompted a reexamination of three mononucleotide-RNase complexes from this laboratory resulting in a re-interpretation of the complex that involved a guanine mononucleotide.

The segmented anisotropic refinement of monoclinic papain by the application of the rigid-body TLS model and comparison to bovine ribonuclease A.

The anisotropic displacements of selected rigid groups in monoclinic papain have been refined from X-ray diffraction data by application of the rigid-body TLS model. The rigid groups chosen were the aromatic side chains of tryptophan, tyrosine, histidine and phenylalanine, and the planar carboxylic and guanidinium side chains of aspartic acid, glutamic acid, glutamine, asparagine and arginine. The derived translation and libration tensors have been compared with those previously derived for bovine ribonuclease A and provide evidence for different modes and anisotropies of displacement over the two proteins.

Domain flexibility in aspartic proteinases.

Comparison of the three-dimensional structures of native endothiapepsin (EC 3.4.23.6) and 15 endothiapepsin oligopeptide inhibitor complexes defined at high resolution by X-ray crystallography shows that endothiapepsin exists in two forms differing in the relative orientation of a domain comprising residues 190-302. There are relatively few interactions between the two parts of the enzyme; consequently, they can move as separate rigid bodies. A translational, librational, and screw analysis of the thermal parameters of endothiapepsin also supports a model in which the two parts can move relative to each other. In the comparison of different aspartic proteinases, the rms values are reduced by up to 47% when the two parts of the structure are superposed independently. This justifies description of the differences, including those between pepsinogen and pepsin (EC 3.4.34.1), as a rigid movement of one part relative to another although considerable distortions within the domains also occur. The consequence of the rigid body movement is a change in the shape of the active site cleft that is largest around the S3 pocket. This is associated with a different position and conformation of the inhibitors that are bound to the two endothiapepsin forms. The relevance of these observations to a model of the hydrolysis by aspartic proteinases is briefly discussed.

Some different strategies of least-squares refinement of a molecule.

Refinements of a macromolecule (ribonuclease-A) based on structure amplitudes, magnitude of F, and structure amplitude squares, magnitude of F2, were carried out and the results compared. Although the conventional R values are higher for the magnitude of F2 refinement, positional parameters from both types of refinement were not significantly different. However, the mean-square displacements from magnitude of F2 refinements were systematically higher than for those using magnitude F. Various resolution windows and weighting schemes were employed during the work. Electron density maps were examined for magnitude of F2 refinements and were very similar to those using magnitude of F in spite of a conventional R factor of 0.29 using all 1.4 A data. While magnitude of F2 refinements may be formally more correct than magnitude of F refinements, there is little evidence that magnitude of F2 refinement is superior provided that a reasonable weighting strategy is adopted.