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E1 and E2 (E1E2), the fusion proteins of Hepatitis C Virus (HCV), are unlike that of any other virus yet described, and the detailed molecular mechanisms of HCV entry/fusion remain unknown. Hypervariable region-1 (HVR-1) of E2 is a putative intrinsically disordered protein tail. Here, we demonstrate that HVR-1 has an autoinhibitory function that suppresses the activity of E1E2 on free virions; this is dependent on its conformational entropy. Thus, HVR-1 is akin to a safety catch that prevents premature triggering of E1E2 activity. Crucially, this mechanism is turned off by host receptor interactions at the cell surface to allow entry. Mutations that reduce conformational entropy in HVR-1, or genetic deletion of HVR-1, turn off the safety catch to generate hyper-reactive HCV that exhibits enhanced virus entry but is thermally unstable and acutely sensitive to neutralising antibodies. Therefore, the HVR-1 safety catch controls the efficiency of virus entry and maintains resistance to neutralising antibodies. This discovery provides an explanation for the ability of HCV to persist in the face of continual immune assault and represents a novel regulatory mechanism that is likely to be found in other viral fusion machinery.

We present flexiMAP (flexible Modeling of Alternative PolyAdenylation), a new beta-regression-based method implemented in R, for discovering differential alternative polyadenylation events in standard RNA-seq data. We show, using both simulated and real data, that flexiMAP exhibits a good balance between specificity and sensitivity and compares favourably to existing methods, especially at low fold changes. In addition, the tests on simulated data reveal some hitherto unrecognised caveats of existing methods. Importantly, flexiMAP allows modeling of multiple known covariates that often confound the results of RNA-seq data analysis. This repository contains scripts and all data required to reproduce data in the manuscript. _simulationData zip files contain extensive set of simulations which should serve as a useful resource for the development of similar methods in the future. Additionally, it contains 3’ sequencing data (PolyA-seq) and RNA-seq data from the Human Brain Reference and the Universal Human Reference MAQC samples (Bullard et al., 2010) that was used in the manuscript., {"references":["Krzysztof J. Szkop, David S. Moss and Irene Nobeli flexiMAP: A regression-based method for discovering differential alternative polyadenylation events in standard RNA-seq data"]}

Over 500 missense F8 mutations have been reported to cause non-severe haemophilia A. Some F8 genotypes appear to confer a higher risk of inhibitor formation than others and individuals with the same F8 genotype may have differing risks of inhibitor formation. We present an in silico strategy demonstrating the heterogeneity of factor VIII (FVIII)-derived antigen presentation whilst identifying patterns of human leucocyte antigen (HLA) peptide binding that might predict future inhibitor risk. A well-validated computational tool, NetMHCII, enabled large-scale comparison of predicted antigen presentation between endogenous, mutated FVIII-derived peptides and wild-type, therapeutic FVIII-derived peptides spanning all F8 missense mutation positions reported to The Haemophilia A Mutation, Structure and Resource Site (HADB). We identify 40 F8 genotypes to be 'low risk' at a 50% inhibitory concentration (IC50 )-binding threshold of 300 nmol/l (P = 0·00005), defined as absence of novel peptide-major histocompatibility complex (MHC) surfaces for all 14 common HLA-DR alleles assessed. Analysing each of the possible 7280 F8 genotype/HLA-DR permutations individually at an IC50 threshold of 300 nmol/l, 65% are predicted to not generate a novel peptide-MHC surface that would be necessary to engage T cell help for subsequent anti-FVIII antibody generation. This study demonstrates the future importance of interpreting F8 genotype in the context of an individual's HLA profile to personalize inhibitor risk prediction.

Highlights • Etx mutant Y30A-Y196A showed markedly reduced cytotoxicity towards MDCK.2 cells. • Y30A-Y196A is inactive in mice after intraperitoneal administration. • Y30A-Y196A is able to induce a specific antibody response in rabbits. • Y30A-Y196A polyclonal antibody is able to induce protective immunity in vitro. • Y30A-Y196A could form the basis of a recombinant vaccine against enterotoxemia., Epsilon toxin (Etx) is a β-pore-forming toxin produced by Clostridium perfringens toxinotypes B and D and plays a key role in the pathogenesis of enterotoxemia, a severe, often fatal disease of ruminants that causes significant economic losses to the farming industry worldwide. This study aimed to determine the potential of a site-directed mutant of Etx (Y30A-Y196A) to be exploited as a recombinant vaccine against enterotoxemia. Replacement of Y30 and Y196 with alanine generated a stable variant of Etx with significantly reduced cell binding and cytotoxic activities in MDCK.2 cells relative to wild type toxin (>430-fold increase in CT50) and Y30A-Y196A was inactive in mice after intraperitoneal administration of trypsin activated toxin at 1000× the expected LD50 dose of trypsin activated wild type toxin. Moreover, polyclonal antibody raised in rabbits against Y30A-Y196A provided protection against wild type toxin in an in vitro neutralisation assay. These data suggest that Y30A-Y196A mutant could form the basis of an improved recombinant vaccine against enterotoxemia.

Recently, a number of broad-spectrum human antibodies binding to the stalk region of influenza A haemagglutinin (HA) have been isolated. As this region tends to develop substitutions at a slower rate than other regions of HA, a vaccine eliciting such antibodies could have a longer effective life. But this begs a question: is the stalk resistant to change even in the face of evolutionary pressure? In this paper, we analysed the known epitopes in the H3 stalk and, utilizing a collection of 3440 sequences, present a novel approach for detecting putative B-cell epitopes in regions such as this, in which mutations occur infrequently. We concluded that there have been periods of activity in the stalk that are consistent with the evolution of antigenic escape. This work casts light on the presence of stalk-binding antibodies in the population as a whole and, through the analysis of antigenically active regions in the stalk, may contribute to the identification of epitopes that are refractive to change and hence useful for vaccine development.

Clostridium perfringens epsilon toxin (Etx) is a pore-forming toxin responsible for a severe and rapidly fatal enterotoxemia of ruminants. The toxin is classified as a category B bioterrorism agent by the U.S. Government Centres for Disease Control and Prevention (CDC), making work with recombinant toxin difficult. To reduce the hazard posed by work with recombinant Etx, we have used a variant of Etx that contains a H149A mutation (Etx-H149A), previously reported to have reduced, but not abolished, toxicity. The three-dimensional structure of H149A prototoxin shows that the H149A mutation in domain III does not affect organisation of the putative receptor binding loops in domain I of the toxin. Surface exposed tyrosine residues in domain I of Etx-H149A (Y16, Y20, Y29, Y30, Y36 and Y196) were mutated to alanine and mutants Y30A and Y196A showed significantly reduced binding to MDCK.2 cells relative to Etx-H149A that correlated with their reduced cytotoxic activity. Thus, our study confirms the role of surface exposed tyrosine residues in domain I of Etx in binding to MDCK cells and the suitability of Etx-H149A for further receptor binding studies. In contrast, binding of all of the tyrosine mutants to ACHN cells was similar to that of Etx-H149A, suggesting that Etx can recognise different cell surface receptors. In support of this, the crystal structure of Etx-H149A identified a glycan (β-octyl-glucoside) binding site in domain III of Etx-H149A, which may be a second receptor binding site. These findings have important implications for developing strategies designed to neutralise toxin activity.

The ultimate aim of the EU-funded ImmunoGrid project is to develop a natural-scale model of the human immune system—that is, one that reflects both the diversity and the relative proportions of the molecules and cells that comprise it—together with the grid infrastructure necessary to apply this model to specific applications in the field of immunology. These objectives present the ImmunoGrid Consortium with formidable challenges in terms of complexity of the immune system, our partial understanding about how the immune system works, the lack of reliable data and the scale of computational resources required. In this paper, we explain the key challenges and the approaches adopted to overcome them. We also consider wider implications for the present ambitious plans to develop natural-scale, integrated models of the human body that can make contributions to personalized health care, such as the European Virtual Physiological Human initiative. Finally, we ask a key question: How long will it take us to resolve these challenges and when can we expect to have fully functional models that will deliver health-care benefits in the form of personalized care solutions and improved disease prevention?

In this paper we undertake an analysis of the antigenicity of influenza A virus hemagglutinin. We developed a novel computational approach to the identification of antigenically active regions and showed that the amino acid substitutions between successive predominant seasonal strains form clusters that are consistent, in terms of both their location and their size, with the properties of B-cell epitopes in general and with those epitopes that have been identified experimentally in influenza A virus hemagglutinin to date. Such an interpretation provides a biologically plausible framework for an understanding of the location of antigenically important substitutions that is more specific than the canonical “antigenic site” model and provides an effective basis for deriving models that predict antigenic escape in the H3N2 subtype. Our results support recent indications that antibodies binding to the “stalk” region of hemagglutinin are found in the human population and exert evolutionary pressure on the virus. Our computational approach provides a possible method for identifying antigenic escape through evolution in this region, which in some cases will not be identified by the hemagglutinin inhibition assay.

We are used to thinking of proteins as beneficial, so it is surprising to realize that the most toxic substances known to man are also protein molecules. These are the bacterial exotoxins, proteins secreted by pathogenic bacteria. Toxicity is measured by the median lethal dose (LD50). An LD50 value is defined as the mass of toxin per kg of body weight required to wipe out half of an animal population. Whereas classic poisons, such as potassium cyanide or arsenic trioxide, have LD50 values in the range 5–15 mg/ kg, the causative agent of botulism, botulinum toxin, has an LD50 in the range 1–3 ng/kg, a million times more toxic!

We have developed a computational Grid that enables us to exploit through a single interface a range of local, national and international resources. It insulates the user as far as possible from issues concerning administrative boundaries, passwords and different operating system features. This work has been undertaken as part of the European Union ImmunoGrid project whose aim is to develop simulations of the immune system at the molecular, cellular and organ levels. The ImmunoGrid consortium has members with computational resources on both sides of the Atlantic. By making extensive use of existing Grid middleware, our Grid has enabled us to exploit consortium and publicly available computers in a unified way, notwithstanding the diverse local software and administrative environments. We took 40 000 polypeptide sequences from 4000 avian and mammalian influenza strains and used a neural network for class I T-cell epitope prediction tools for 120 class I alleles and haplotypes to generate over 14 million high-quality protein–peptide binding predictions that we are mapping onto the three-dimensional structures of the proteins. By contrast, the Grid is also being used for developing new methods for class T-cell epitope predictions, where we have running batches of 120 molecular dynamics free-energy calculations.

Epitopes mediated by T cells lie at the heart of the adaptive immune response and form the essential nucleus of anti-tumour peptide or epitope-based vaccines. Antigenic T cell epitopes are mediated by major histocompatibility complex (MHC) molecules, which present them to T cell receptors. Calculating the affinity between a given MHC molecule and an antigenic peptide using experimental approaches is both difficult and time consuming, thus various computational methods have been developed for this purpose. A server has been developed to allow a structural approach to the problem by generating specific MHC:peptide complex structures and providing configuration files to run molecular modelling simulations upon them. A system has been produced which allows the automated construction of MHC:peptide structure files and the corresponding configuration files required to execute a molecular dynamics simulation using NAMD. The system has been made available through a web-based front end and stand-alone scripts. Previous attempts at structural prediction of MHC:peptide affinity have been limited due to the paucity of structures and the computational expense in running large scale molecular dynamics simulations. The MHCsim server (http://igrid-ext.cryst.bbk.ac.uk/MHCsim) allows the user to rapidly generate any desired MHC:peptide complex and will facilitate molecular modelling simulation of MHC complexes on an unprecedented scale.

Antigenic peptide is presented to a T-cell receptor through the formation of a stable complex with a Major Histocompatibility Complex (MHC) molecule. Various predictive algorithms have been developed to estimate a peptide’s capacity to form a stable complex with a given MHC Class II allele, a technique integral to the strategy of vaccine design. These have previously incorporated such computational techniques as quantitative matrices and neural networks. We have developed a novel predictive technique that uses molecular modeling of predetermined crystal structures to estimate the stability of an MHC Class II peptide complex. This is the 1st structure-based technique, as previous methods have been based on binding data. ROC curves are used to quantify the accuracy of the molecular modeling technique. The novel predictive technique is found to be comparable with the best predictive software currently available.

The extent of the role of N-linked glycans (N-glycans) in shielding influenza A hemagglutinin (HA) against host antibodies has proved controversial, with different authors making widely different assumptions. One common assumption is that N-glycans physically shield surface residues that are near to glycosylation sites, thereby preventing antibodies from binding to them. However, it is unclear, from existing experimental evidence, whether antibodies that bind close to N-glycans are a rare or commonplace feature of human herd immune responses to influenza AHA. The aim of this paper is to present a computational analysis of mutations in the vicinity of N-glycans that will facilitate a better understanding of their protective role. We identify, from an analysis of over 6000 influenza A H3N2 sequences, a set of residues adjacent to N-glycosylation sites that are highly likely to be involved in antigenic escape from host antibodies. Fifteen of these residues occur within 10 A of an N-glycosylation site. Hence, we conclude that it is relatively common for antibodies to bind in close proximity to N-glycans on the surface ofHA, with any shielding effect largely attributable to the inability of host antibodies to bind across an N-glycan attachment site, rather than to the physical masking of neighboring residues.

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 Contact: d.moss@mail.cryst.bbk.ac.uk; m.williams@biochemistry.ucl.ac.uk * To whom correspondence should be addressed. 3 Present address: Celltech Chiroscience, Granta Park, Great Abingdon, Cambridge CB1 6GS, UK. 4 Present address: Department of Biochemistry & Molecular Biology, University College London, Gower Street, London WC1E 6BT, UK.

Differences in the biological properties of the Clostridium perfringens phospholipase C (alpha-toxin) and the C. bifermentans phospholipase C (Cbp) have been attributed to differences in their carboxy-terminal domains. Three residues in the carboxy-terminal domain of alpha-toxin, which have been proposed to play a role in membrane recognition (D269, Y331 and F334), are not conserved in Cbp (Y, L and I respectively). We have characterised D269Y, Y331L and F334I variant forms of alpha-toxin. Variant D269Y had reduced phospholipase C activity towards aggregated egg yolk phospholipid but increased haemolytic and cytotoxic activity. Variants Y331L and F334I showed a reduction in phospholipase C, haemolytic and cytotoxic activities indicating that these substitutions contribute to the reduced haemolytic and cytotoxic activity of Cbp.

Background: Clostridium perfringens toxin NetB is a key factor in avian necrotic enteritis. Results: NetB forms heptameric pores structurally similar to Staphylococcus aureus toxins but lacks a phosphocholine binding pocket. NetB activity is enhanced by cholesterol. Conclusion: NetB has distinct binding specificity, and cholesterol may act as a receptor. Significance: The structure of NetB will facilitate development of control measures against necrotic enteritis., NetB is a pore-forming toxin produced by Clostridium perfringens and has been reported to play a major role in the pathogenesis of avian necrotic enteritis, a disease that has emerged due to the removal of antibiotics in animal feedstuffs. Here we present the crystal structure of the pore form of NetB solved to 3.9 Å. The heptameric assembly shares structural homology to the staphylococcal α-hemolysin. However, the rim domain, a region that is thought to interact with the target cell membrane, shows sequence and structural divergence leading to the alteration of a phosphocholine binding pocket found in the staphylococcal toxins. Consistent with the structure we show that NetB does not bind phosphocholine efficiently but instead interacts directly with cholesterol leading to enhanced oligomerization and pore formation. Finally we have identified conserved and non-conserved amino acid positions within the rim loops that significantly affect binding and toxicity of NetB. These findings present new insights into the mode of action of these pore-forming toxins, enabling the design of more effective control measures against necrotic enteritis and providing potential new tools to the field of bionanotechnology.

Clostridium perfringens epsilon toxin (Etx) is a pore-forming toxin responsible for a severe and rapidly fatal enterotoxemia of ruminants. The toxin is classified as a category B bioterrorism agent by the U.S. Government Centres for Disease Control and Prevention (CDC), making work with recombinant toxin difficult. To reduce the hazard posed by work with recombinant Etx, we have used a variant of Etx that contains a H149A mutation (Etx-H149A), previously reported to have reduced, but not abolished, toxicity. The three-dimensional structure of H149A prototoxin shows that the H149A mutation in domain III does not affect organisation of the putative receptor binding loops in domain I of the toxin. Surface exposed tyrosine residues in domain I of Etx-H149A (Y16, Y20, Y29, Y30, Y36 and Y196) were mutated to alanine and mutants Y30A and Y196A showed significantly reduced binding to MDCK.2 cells relative to Etx-H149A that correlated with their reduced cytotoxic activity. Thus, our study confirms the role of surface exposed tyrosine residues in domain I of Etx in binding to MDCK cells and the suitability of Etx-H149A for further receptor binding studies. In contrast, binding of all of the tyrosine mutants to ACHN cells was similar to that of Etx-H149A, suggesting that Etx can recognise different cell surface receptors. In support of this, the crystal structure of Etx-H149A identified a glycan (β-octyl-glucoside) binding site in domain III of Etx-H149A, which may be a second receptor binding site. These findings have important implications for developing strategies designed to neutralise toxin activity.

Background Protein structures provide a valuable resource for rational drug design. For a protein with no known ligand, computational tools can predict surface pockets that are of suitable size and shape to accommodate a complementary small-molecule drug. However, pocket prediction against single static structures may miss features of pockets that arise from proteins' dynamic behaviour. In particular, ligand-binding conformations can be observed as transiently populated states of the apo protein, so it is possible to gain insight into ligand-bound forms by considering conformational variation in apo proteins. This variation can be explored by considering sets of related structures: computationally generated conformers, solution NMR ensembles, multiple crystal structures, homologues or homology models. It is non-trivial to compare pockets, either from different programs or across sets of structures. For a single structure, difficulties arise in defining particular pocket's boundaries. For a set of conformationally distinct structures the challenge is how to make reasonable comparisons between them given that a perfect structural alignment is not possible. Results We have developed a computational method, Provar, that provides a consistent representation of predicted binding pockets across sets of related protein structures. The outputs are probabilities that each atom or residue of the protein borders a predicted pocket. These probabilities can be readily visualised on a protein using existing molecular graphics software. We show how Provar simplifies comparison of the outputs of different pocket prediction algorithms, of pockets across multiple simulated conformations and between homologous structures. We demonstrate the benefits of use of multiple structures for protein-ligand and protein-protein interface analysis on a set of complexes and consider three case studies in detail: i) analysis of a kinase superfamily highlights the conserved occurrence of surface pockets at the active and regulatory sites; ii) a simulated ensemble of unliganded Bcl2 structures reveals extensions of a known ligand-binding pocket not apparent in the apo crystal structure; iii) visualisations of interleukin-2 and its homologues highlight conserved pockets at the known receptor interfaces and regions whose conformation is known to change on inhibitor binding. Conclusions Through post-processing of the output of a variety of pocket prediction software, Provar provides a flexible approach to the analysis and visualization of the persistence or variability of pockets in sets of related protein structures.

Clostridium perfringens enterotoxin (CPE) is a major cause of food poisoning and antibiotic-associated diarrhea. Upon its release from C. perfringens spores, CPE binds to its receptor, claudin, at the tight junctions between the epithelial cells of the gut wall and subsequently forms pores in the cell membranes. A number of different complexes between CPE and claudin have been observed, and the process of pore formation has not been fully elucidated. We have determined the three-dimensional structure of the soluble form of CPE in two crystal forms by X-ray crystallography, to a resolution of 2.7 and 4.0 A, respectively, and found that the N-terminal domain shows structural homology with the aerolysin-like β-pore-forming family of proteins. We show that CPE forms a trimer in both crystal forms and that this trimer is likely to be biologically relevant but is not the active pore form. We use these data to discuss models of pore formation.

Motivation: Modelling antigenic shift in influenza A H3N2 can help to predict the efficiency of vaccines. The virus is known to exhibit sudden jumps in antigenic distance, and prediction of such novel strains from amino acid sequence differences remains a challenge. Results: From analysis of 6624 amino acid sequences of wild-type H3, we propose updates to the frequently referenced list of 131 amino acids located at or near the five identified antibody binding regions in haemagglutinin (HA). We introduce a class of predictive models based on the analysis of amino acid changes in these binding regions, and extend the principle to changes in HA1 as a whole by dividing the molecule into regional bands. Our results show that a range of simple models based on banded changes give better predictive performance than models based on the established five canonical regions and can identify a higher proportion of vaccine escape candidates among novel strains than a current state-of-the-art model. Contact: wlees01@mail.cryst.bbk.ac.uk Supplementary information: Supplementary Data is available at Bioinformatics online.

International audience; IMGT, the international ImMunoGeneTics information system (http://imgt.cines.fr), is the reference in immunogenetics and immunoinformatics. IMGT standardizes and manages the complex immunogenetic data that include the immunoglobulins (IG) or antibodies, the T cell receptors (TR), the major histocompatibility complex (MHC) and the related proteins of the immune system (RPI), which belong to the immunoglobulin superfamily (IgSF) and the MHC superfamily (MhcSF). The accuracy and consistency of IMGT data and the coherence between the different IMGT components (databases, tools and Web resources) are based on IMGT-ONTOLOGY, the first ontology for immunogenetics and immunoinformatics. IMGT-ONTOLOGY manages the immunogenetics knowledge through diverse facets relying on seven axioms, 'IDENTIFICATION', 'DESCRIPTION', 'CLASSIFICATION', 'NUMEROTATION', 'LOCALIZATION', 'ORIENTATION' and 'OBTENTION', that postulate that objects, processes and relations have to be identified, described, classified, numerotated, localized, orientated, and that the way they are obtained has to be determined. These axioms constitute the Formal IMGT-ONTOLOGY, also designated as IMGT-Kaleidoscope. These axioms have been essential for the conceptualization of the molecular immunogenetics knowledge and for the creation of IMGT. Indeed all the components of the IMGT integrated system have been developed, based on standardized concepts and relations, thus allowing IMGT to bridge biological and computational spheres in bioinformatics. The same axioms can be used to generate concepts for multi-scale level approaches at the molecule, cell, tissue, organ, organism or population level, emphasizing the generalization of the application domain. In that way the Formal IMGT-ONTOLOGY represents a paradigm for the elaboration of ontologies in system biology.

The measurement of the longitudinal speed of sound in crystals of ribonuclease-A and in human haemoglobin using laser-generated ultrasound is reported. Average values of 1784 (72) m s-1 and 1828 m s-1 were obtained. As a control the speed of sound transmitted through a compacted disc of lysozyme powder was measured as 2004(23)ms-1. The measured longitudinal acoustic velocities and the transverse velocity, estimated from a knowledge of Young's modulus, were used to estimate the acoustic contribution to the mean-square displacements of protein molecules as determined by X-ray crystallography. It was found that thermally induced acoustic vibrations make a significant contribution to the total atomic disorder, estimated to be in the range 0.04-0.11 A2 for the proteins studied. Such single-crystal estimates are required for calculation of the acoustic component of the diffuse scattering in protein crystal X-ray diffraction.

Epitopes mediated by T cells lie at the heart of the adaptive immune response and form the essential nucleus of anti-tumour peptide or epitope-based vaccines. Antigenic T cell epitopes are mediated by major histocompatibility complex (MHC) molecules, which present them to T cell receptors. Calculating the affinity between a given MHC molecule and an antigenic peptide using experimental approaches is both difficult and time consuming, thus various computational methods have been developed for this purpose. A server has been developed to allow a structural approach to the problem by generating specific MHC:peptide complex structures and providing configuration files to run molecular modelling simulations upon them. A system has been produced which allows the automated construction of MHC:peptide structure files and the corresponding configuration files required to execute a molecular dynamics simulation using NAMD. The system has been made available through a web-based front end and stand-alone scripts. Previous attempts at structural prediction of MHC:peptide affinity have been limited due to the paucity of structures and the computational expense in running large scale molecular dynamics simulations. The MHCsim server (http://igrid-ext.cryst.bbk.ac.uk/MHCsim) allows the user to rapidly generate any desired MHC:peptide complex and will facilitate molecular modelling simulation of MHC complexes on an unprecedented scale.

Neutralising antibodies (inhibitors) are increasingly recognized to be a life-time risk in non-severe hemophilia A patients exposed to factor VIII concentrates. It is currently not possible to reliably identify the variable inhibitor risk between individuals. Risk appears to differ between F8 genotypes but also for individuals living with the same F8 genotype. As a T cell dependent process, the wild-type, factor concentrate-derived peptide sequences spanning the F8 mutation position are presented by class II MHC and responsible for driving the T helper response and subsequent B cell response. We hypothesize that the primary sequences of the 20,469 proteins in the human proteome will, coincidentally, contain short primary sequences homologous to key immunogenic peptides derived from the therapeutic factor VIII. We present in silico data and correlation with published registry inhibitor data (Fisher's exact test) to demonstrate the potential impact of such "proteome protection" and future potential to more reliably stratify individuals between low/negligible risk and more significant risk of inhibitor formation. We utilize a well-validated, computational tool, NetMHC-II, to enable large scale, computational comparison of predicted antigen presentation between endogenous, mutated FVIII derived peptides and factor-concentrate derived, wild-type FVIII peptides spanning all 520 F8 missense mutations listed on www.hadb.org. NetMHC-II analyses peptide presentation by 14 class II MHC HLA-DR alleles, resulting in analysis of 7,280 (520 x 14) permutations of F8 -MHC-II. We identify 56% (n=4,077) of these permutations to be at low/negligible risk of inhibitor formation, at a binding threshold of 500nM, defined as absence of a novel peptide-MHC surface capable of driving a helper T cell response (p=0.005). When cross referenced with potential homologous sequences buried anywhere in the human proteome (http://www.ebi.ac.uk/reference_proteomes), a further 1,237 F8 -MHC-II combinations are afforded "proteome protection" due to direct sequence homology between FVIII-derived peptide and peptide(s) derived from other proteins. This increases the total number of F8 -MHC-II combinations predicted to be unable to drive a T cell response to 73% (n=5,314). The residual 1,966 (27%) F8 -MHC-II combinations are predicted to retain the ability to present novel-interface FVIII-derived peptides to T cells with an IC50 Previous work exploring in silico prediction of FVIII-derived peptide presentation may have overestimated the number of F8 -MHC II combinations deemed to be at risk of contributing to inhibitor formation. Our data suggests an additional mechanism "protecting" a larger proportion of those living with non-severe HA from inhibitor formation. The contribution of "proteome protection" further reduces the "at risk" F8 -MHC II permutations to be more representative of clinically observed inhibitor rates. We identify a potential novel tolerance mechanism and provide key data for future in vitro validation strategies. Disclosures No relevant conflicts of interest to declare.

Background MHC Class I molecules present antigenic peptides to cytotoxic T cells, which forms an integral part of the adaptive immune response. Peptides are bound within a groove formed by the MHC heavy chain. Previous approaches to MHC Class I-peptide binding prediction have largely concentrated on the peptide anchor residues located at the P2 and C-terminus positions. Results A large dataset comprising MHC-peptide structural complexes was created by re-modelling pre-determined x-ray crystallographic structures. Static energetic analysis, following energy minimisation, was performed on the dataset in order to characterise interactions between bound peptides and the MHC Class I molecule, partitioning the interactions within the groove into van der Waals, electrostatic and total non-bonded energy contributions. Conclusion The QSAR techniques of Genetic Function Approximation (GFA) and Genetic Partial Least Squares (G/PLS) algorithms were used to identify key interactions between the two molecules by comparing the calculated energy values with experimentally-determined BL50 data. Although the peptide termini binding interactions help ensure the stability of the MHC Class I-peptide complex, the central region of the peptide is also important in defining the specificity of the interaction. As thermodynamic studies indicate that peptide association and dissociation may be driven entropically, it may be necessary to incorporate entropic contributions into future calculations.

Epsilon-toxin from Clostridium perfringens is a lethal toxin. Recent studies suggest that the toxin acts via an unusually potent pore-forming mechanism. Here we report the crystal structure of epsilon-toxin, which reveals structural similarity to aerolysin from Aeromonas hydrophila. Pore-forming toxins can change conformation between soluble and transmembrane states. By comparing the two toxins, we have identified regions important for this transformation.

The collaborative computing project in biosequence and structure analysis (CCPII) was established to foster bioinformatics in the broad community and the UK research community in particular. A World-Wide Web site called 'The Bioinformatics Resource' has been created containing a comprehensive set of information resources of use to the bioinformatician. The activities of CCPII are complementary to other providers of molecular biology information such as the BIOSCI electronic communication forum, which was established to facilitate communication between professionals in the biological sciences.

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.

The Internet is becoming an important medium for the delivery of educational materials. However, relatively few institutions are delivering whole courses using this medium. More often, the technologies are used to complement traditional courses, which may be given face-to-face or at a distance (Farrell, 1999). The Department of Crystallography at Birkbeck College, London, has been in the vanguard of the development of 'virtual education', providing some of the first accredited postgraduate courses in the UK to be offered entirely using the new technologies. For the past four years, we have been running an Advanced Certificate course entitled 'Principles of Protein Structure using the Internet'1 (Sansom, Walshaw and Moss, 1997) (PPS). See http://www.cryst.bbk.aauk/pps for more details. This was one of the first tutor-assisted, accredited, university-level courses to be taught entirely over the Internet, and is certainly the first in biochemistry in the UK.DOI:10.1080/0968776000080204

Background: Little is known of the intermolecular organization of crystallins in the protein-packed eye lens. The tetrameric structure of the 200 000 Da avian δ -crystallin, which is closely related to the enzyme argininosuccinate lyase and is characteristic of the accommodating, soft lens of birds, has recently been solved at atomic resolution at acidic pH. To help understand how δ -crystallin remains soluble at the very high concentrations found in the avian lens we have now crystallized turkey δ -crystallin at around neutral pH and examined its intermolecular interactions. Results Turkey δ -crystallin has been crystallized around neutral pH. The X-ray structure has been solved at 4.5 a resolution in space group C2 with three and a half tetramers in the asymmetric unit. The symmetrical 222 tetramers have a novel packing arrangement consisting of continuous helices, with 7 3 2 non-crystallographic symmetry, in an approximately hexagonal close-packed array. The internal 222 symmetry of the tetramers allows different polymeric chains to be constructed, based on the tetramer–tetramer association observed in the crystalline helix. It is possible to build a model of a tubule of diameter 212 a that is very similar to observed tubules of bovine argininosuccinate lyase. Conclusion Elements of helical organization may occur in the concentrated solution of the avian eye lens where δ -crystallin is the prominent protein. The symmetry of the tetramer provides a choice in the direction of growth of a helix at each link so that highly hydrated irregular polymers may be formed rather than large compact regular structures that would not be compatible with a transparent lens.

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.

Background G protein-coupled receptors (GPCRs) play important physiological roles transducing extracellular signals into intracellular responses. Approximately 50% of all marketed drugs target a GPCR. There remains considerable interest in effectively predicting the function of a GPCR from its primary sequence. Findings Using techniques drawn from data mining and proteochemometrics, an alignment-free approach to GPCR classification has been devised. It uses a simple representation of a protein's physical properties. GPCRTree, a publicly-available internet server, implements an algorithm that classifies GPCRs at the class, sub-family and sub-subfamily level. Conclusion A selective top-down classifier was developed which assigns sequences within a GPCR hierarchy. Compared to other publicly available GPCR prediction servers, GPCRTree is considerably more accurate at every level of classification. The server has been available online since March 2008 at URL: http://igrid-ext.cryst.bbk.ac.uk/gpcrtree/.

Background pKa values are a measure of the protonation of ionizable groups in proteins. Ionizable groups are involved in intra-protein, protein-solvent and protein-ligand interactions as well as solubility, protein folding and catalytic activity. The pKa shift of a group from its intrinsic value is determined by the perturbation of the residue by the environment and can be calculated from three-dimensional structural data. Results Here we use a large dataset of experimentally-determined pKas to analyse the performance of different prediction techniques. Our work provides a benchmark of available software implementations: MCCE, MEAD, PROPKA and UHBD. Combinatorial and regression analysis is also used in an attempt to find a consensus approach towards pKa prediction. The tendency of individual programs to over- or underpredict the pKa value is related to the underlying methodology of the individual programs. Conclusion Overall, PROPKA is more accurate than the other three programs. Key to developing accurate predictive software will be a complete sampling of conformations accessible to protein structures.

A least-squares refinement program RESTRAIN has been developed, which is capable of refining macromolecular structures using structure amplitudes, phases from isomorphous replacement or anomalous scattering and pseudo-energy restraints. In addition to positional parameters and isotropic temperature factors, anisotropic mean-square displacements may be refined either as individual atomic U tensors or as TLS tensors applied to groups of atoms. Anharmonic effects may be handled by coupling together occupancies to enable the electron density of an atomic group to be distributed over more than one subsite. A novel way of restraining groups of atoms to be planar has been developed that does not require dummy atoms and does not restrain the plane to lie in its current orientation.

The three-dimensional structure of the eye lens protein, bovine gamma-crystallin II, has been determined at 2.6 A resolution. The protein has a tow domain beta-structure, folded into four remarkably similar 'Greed key' motifs, and shows the highest internal symmetry of any protein studied by X-ray analysis. Although the symmetrical structure seems very stable, the arrangement of the sulphydryl groups would allow intramolecular cross-linking leading to possible destabilization, and intermolecular cross-linking leading to aggregation, both of which may be important to cataract formation.

The anisotropic displacements of selected rigid groups in bovine ribonuclease A have been refined from X-ray diffraction data by the application of the rigid-body TLS model. The rigid groups chosen were the side chains of tyrosine, histidine and phenylalanine and the planar side chains of aspartic acid, glutamic acid, glutamine, asparagine and arginine. The method has also been applied to the co-crystallizing active-site sulfate anion. This has enabled the description of the motion of the above-mentioned side-chain atoms by anisotropic displacement ellipsoids from a 1.45 A refinement. The hydrophobic side groups in the protein core show mainly translational motion, with mean-square librations of 20 deg2 which are similar to those found in some close-packed crystals of small organic molecules. Librational displacements are much more significant in the hydrophilic side groups where their magnitudes can be correlated with solvent accessibility. Large liberations of some solvent exposed side chains correspond with the breakdown of a simple TLS model and the existence of multiple orientations of the side groups. The TLS model has also been applied to the whole protein molecule and shows that the average motion is approximately isotropic with little librational character.