A site for direct integrin αvß6·uPAR interaction from structural modelling and docking.

Integrin αvß6 is an epithelially-restricted heterodimeric transmembrane glycoprotein, known to interact with the urokinase plasminogen activating receptor (uPAR), playing a critical role in cancer progression. While the X-ray crystallographic structures of segments of other integrin heterodimers are known, there is no structural information for the complete αvß6 integrin to assess its direct interaction with uPAR. We have performed structural analysis of αvß6·uPAR interactions using model data with docking simulations to pinpoint their interface, in accord with earlier reports of the ß-propeller region of integrin α-chain interacting with uPAR. Interaction of αvß6·uPAR was demonstrated by our previous study using immunoprecipitation coupled with proteomic analysis by mass spectrometry. Recently this interaction was validated with proximity ligation assays and peptide arrays. The data suggested that two potential peptide regions from domain II and one peptide region from domain III of uPAR, interact with αvß6 integrin. Only the peptide region from domain III is consistent with the three-dimensional interaction site proposed in this study. The molecular basis of integrin αvß6·uPAR binding using structural data is discussed for its implications as a potential therapeutic target in cancer management.

Unlocking the puzzling biology of the black Périgord truffle Tuber melanosporum.

The black Périgord truffle (Tuber melanosporum Vittad.) is a highly prized food today, with its unique scent (i.e., perfume) and texture. Despite these attributes, it remains relatively poorly studied, lacking "omics" information to characterize its biology and biochemistry, especially changes associated with freshness and the proteins/metabolites responsible for its organoleptic properties. In this study, we have functionally annotated the truffle proteome from the 2010 T. melanosporum genome comprising 12,771 putative nonredundant proteins. Using sequential BLAST search strategies, we identified homologues for 2587 proteins with 2486 (96.0%) fungal homologues (available from http://biolinfo.org/protannotator/blacktruffle.php). A combined 1D PAGE and high-accuracy LC-MS/MS proteomic study was employed to validate the results of the functional annotation and identified 836 (6.5%) proteins, of which 47.5% (i.e., 397) were present in our bioinformatics studies. Our study, functionally annotating 6487 black Périgord truffle proteins and confirming 836 by proteomic experiments, is by far the most comprehensive study to date contributing significantly to the scientific community. This study has resulted in the functional characterization of novel proteins to increase our biological understanding of this organism and to uncover potential biomarkers of authenticity, freshness, and perfume maturation.

Understanding TR binding to pMHC complexes: how does a TR scan many pMHC complexes yet preferentially bind to one.

Understanding the basis of the binding of a T cell receptor (TR) to the peptide-MHC (pMHC) complex is essential due to the vital role it plays in adaptive immune response. We describe the use of computed binding (free) energy (BE), TR paratope, pMHC epitope, molecular surface electrostatic potential (MSEP) and calculated TR docking angle (θ) to analyse 61 TR/pMHC crystallographic structures to comprehend TR/pMHC interaction. In doing so, we have successfully demonstrated a novel/rational approach for θ calculation, obtained a linear correlation between BE and θ without any "codon" or amino acid preference, provided an explanation for TR ability to scan many pMHC ligands yet specifically bind one, proposed a mechanism for pMHC recognition by TR leading to T cell activation and illustrated the importance of the peptide in determining TR specificity, challenging the "germline bias" theory.

MPID-T2: a database for sequence-structure-function analyses of pMHC and TR/pMHC structures.

UNLABELLED: Sequence-structure-function information is critical in understanding the mechanism of pMHC and TR/pMHC binding and recognition. A database for sequence-structure-function information on pMHC and TR/pMHC interactions, MHC-Peptide Interaction Database-TR version 2 (MPID-T2), is now available augmented with the latest PDB and IMGT/3Dstructure-DB data, advanced features and new parameters for the analysis of pMHC and TR/pMHC structures. AVAILABILITY: http://biolinfo.org/mpid-t2. CONTACT: shoba.ranganathan@mq.edu.au SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

A comparative structural bioinformatics analysis of inherited mutations in ß-D-Mannosidase across multiple species reveals a genotype-phenotype correlation.

BACKGROUND: Lysosomal ß-D-mannosidase is a glycosyl hydrolase that breaks down the glycosidic bonds at the non-reducing end of N-linked glycoproteins. Hence, it is a crucial enzyme in polysaccharide degradation pathway. Mutations in the MANBA gene that codes for lysosomal ß-mannosidase, result in improper coding and malfunctioning of protein, leading to ß-mannosidosis. Studying the location of mutations on the enzyme structure is a rational approach in order to understand the functional consequences of these mutations. Accordingly, the pathology and clinical manifestations of the disease could be correlated to the genotypic modifications. RESULTS: The wild-type and inherited mutations of ß-mannosidase were studied across four different species, human, cow, goat and mouse employing a previously demonstrated comprehensive homology modeling and mutational mapping technique, which reveals a correlation between the variation of genotype and the severity of phenotype in ß-mannosidosis. X-ray crystallographic structure of ß-mannosidase from Bacteroides thetaiotaomicron was used as template for 3D structural modeling of the wild-type enzymes containing all the associated ligands. These wild-type models subsequently served as templates for building mutational structures. Truncations account for approximately 70% of the mutational cases. In general, the proximity of mutations to the active site determines the severity of phenotypic expressions. Mapping mutations to the MANBA gene sequence has identified five mutational hot-spots. CONCLUSION: Although restrained by a limited dataset, our comprehensive study suggests a genotype-phenotype correlation in ß-mannosidosis. A predictive approach for detecting likely ß-mannosidosis is also demonstrated where we have extrapolated observed mutations from one species to homologous positions in other organisms based on the proximity of the mutations to the enzyme active site and their co-location from different organisms. Apart from aiding the detection of mutational hotspots in the gene, where novel mutations could be disease-implicated, this approach also provides a way to predict new disease mutations. Higher expression of the exoglycosidase chitobiase is said to play a vital role in determining disease phenotypes in human and mouse. A bigger dataset of inherited mutations as well as a parallel study of ß-mannosidase and chitobiase activities in prospective patients would be interesting to better understand the underlying reasons for ß-mannosidosis.

pDOCK: a new technique for rapid and accurate docking of peptide ligands to Major Histocompatibility Complexes.

BACKGROUND: Identification of antigenic peptide epitopes is an essential prerequisite in T cell-based molecular vaccine design. Computational (sequence-based and structure-based) methods are inexpensive and efficient compared to experimental approaches in screening numerous peptides against their cognate MHC alleles. In structure-based protocols, suited to alleles with limited epitope data, the first step is to identify high-binding peptides using docking techniques, which need improvement in speed and efficiency to be useful in large-scale screening studies. We present pDOCK: a new computational technique for rapid and accurate docking of flexible peptides to MHC receptors and primarily apply it on a non-redundant dataset of 186 pMHC (MHC-I and MHC-II) complexes with X-ray crystal structures. RESULTS: We have compared our docked structures with experimental crystallographic structures for the immunologically relevant nonameric core of the bound peptide for MHC-I and MHC-II complexes. Primary testing for re-docking of peptides into their respective MHC grooves generated 159 out of 186 peptides with Cα RMSD of less than 1.00 Å, with a mean of 0.56 Å. Amongst the 25 peptides used for single and variant template docking, the Cα RMSD values were below 1.00 A for 23 peptides. Compared to our earlier docking methodology, pDOCK shows upto 2.5 fold improvement in the accuracy and is ~60% faster. Results of validation against previously published studies represent a seven-fold increase in pDOCK accuracy. CONCLUSIONS: The limitations of our previous methodology have been addressed in the new docking protocol making it a rapid and accurate method to evaluate pMHC binding. pDOCK is a generic method and although benchmarks against experimental structures, it can be applied to alleles with no structural data using sequence information. Our outcomes establish the efficacy of our procedure to predict highly accurate peptide structures permitting conformational sampling of the peptide in MHC binding groove. Our results also support the applicability of pDOCK for in silico identification of promiscuous peptide epitopes that are relevant to higher proportions of human population with greater propensity to activate T cells making them key targets for the design of vaccines and immunotherapies.

A multi-species comparative structural bioinformatics analysis of inherited mutations in alpha-D-mannosidase reveals strong genotype-phenotype correlation.

BACKGROUND: Lysosomal alpha-mannosidase is an enzyme that acts to degrade N-linked oligosaccharides and hence plays an important role in mannose metabolism in humans and other mammalian species, especially livestock. Mutations in the gene (MAN2B1) encoding lysosomal alpha-D-mannosidase cause improper coding, resulting in dysfunctional or non-functional protein, causing the disease alpha-mannosidosis. Mapping disease mutations to the structure of the protein can help in understanding the functional consequences of these mutations and thus indirectly, the finer aspects of the pathology and clinical manifestations of the disease, including phenotypic severity as a function of the genotype. RESULTS: A comprehensive homology modeling study of all the wild-type and inherited mutations of lysosomal alpha-mannosidase in four different species, human, cow, cat and guinea pig, reveals a significant correlation between the severity of the genotype and the phenotype in alpha-mannosidosis. We used the X-ray crystallographic structure of bovine lysosomal alpha-mannosidase as template, containing only two disulphide bonds and some ligands, to build structural models of wild-type structures with four disulfide linkages and all bound ligands. These wild-type models were then used as templates for disease mutations. All the truncations and substitutions involving the residues in and around the active site and those that destabilize the fold led to severe genotypes resulting in lethal phenotypes, whereas the mutations lying away from the active site were milder in both their genotypic and phenotypic expression. CONCLUSION: Based on the co-location of mutations from different organisms and their proximity to the enzyme active site, we have extrapolated observed mutations from one species to homologous positions in other organisms, as a predictive approach for detecting likely alpha-mannosidosis. Besides predicting new disease mutations, this approach also provides a way for detecting mutation hotspots in the gene, where novel mutations could be implicated in disease. The current study has identified five mutational hot-spot regions along the MAN2B1 gene. Structural mapping can thus provide a rational approach for predicting the phenotype of a disease, based on observed genotypic variations.