Development of a generic, structural bioinformatics information management system and its application to variation in foot-and-mouth disease virus proteins

Structural biology forms the basis of all functions in an organism from how enzymes work to how a cell is assembled. In silico structural biology has been a rather isolated domain due to the perceived difficulty of working with the tools. This work focused on constructing a web-based Functional Genomics Information Management System (FunGIMS) that will provide biologists access to the most commonly used structural biology tools without the need to learn program or operating specific syntax. The system was designed using a Model-View-Controller architecture which is easy to maintain and expand. It is Python-based with various other technologies incorporated. The specific focus of this work was the Structural module which allows a user to work with protein structures. The database behind the system is based on a modified version of the Macromolecular Structure Database from the EBI. The Structural module provides functionality to explore protein structures at each level of complexity through an easy-to-use interface. The module also provides some analysis tools which allows the user to identify features on a protein sequence as well as to identify unknown protein sequences. Another vital functionality allows the users to build protein models. The user can choose between building models online on downloading a generated script. Similar script generation utilities are provided for mutation modeling and molecular dynamics. A search functionality was also provided which allows the user to search for a keyword in the database. The system was used on three examples in Foot-and-Mouth Disease Virus (FMDV). In the first case, several FMDV proteomes were reannotated and compared to elucidate any functional differences between them. The second case involved the modeling of two FMDV proteins involved in replication, 3C and 3D. Variation between the several different strains were mapped to the structures to understand how variation affects enzymes structure. The last example involved capsid protein stability differences between two subtypes. Models were built and molecular dynamics simulations were run to determine at which protein structure level stability was influenced by the differences between the subtypes. This work provides an important introductory tool for biologists to structural biology.Thesis (PhD)--University of Pretoria, 2009.Biochemistryunrestricte

The BID Domain of Type IV Secretion Substrates Forms a Conserved Four-Helix Bundle Topped with a Hook

The BID (Bep intracellular delivery) domain functions as secretion signal in a subfamily of protein substrates of bacterial type IV secretion (T4S) systems. It mediates transfer of (1) relaxases and the attached DNA during bacterial conjugation, and (2) numerous Bartonella effector proteins (Beps) during protein transfer into host cells infected by pathogenic Bartonella species. Furthermore, BID domains of Beps have often evolved secondary effector functions within host cells. Here, we provide crystal structures for three representative BID domains and describe a novel conserved fold characterized by a compact, antiparallel four-helix bundle topped with a hook. The conserved hydrophobic core provides a rigid scaffold to a surface that, despite a few conserved exposed residues and similarities in charge distribution, displays significant variability. We propose that the genuine function of BID domains as T4S signal may primarily depend on their rigid structure, while the plasticity of their surface may facilitate adaptation to secondary effector functions

The Catalytic Site Atlas 2.0: cataloging catalytic sites and residues identified in enzymes.

Understanding which are the catalytic residues in an enzyme and what function they perform is crucial to many biology studies, particularly those leading to new therapeutics and enzyme design. The original version of the Catalytic Site Atlas (CSA) (http://www.ebi.ac.uk/thornton-srv/databases/CSA) published in 2004, which catalogs the residues involved in enzyme catalysis in experimentally determined protein structures, had only 177 curated entries and employed a simplistic approach to expanding these annotations to homologous enzyme structures. Here we present a new version of the CSA (CSA 2.0), which greatly expands the number of both curated (968) and automatically annotated catalytic sites in enzyme structures, utilizing a new method for annotation transfer. The curated entries are used, along with the variation in residue type from the sequence comparison, to generate 3D templates of the catalytic sites, which in turn can be used to find catalytic sites in new structures. To ease the transfer of CSA annotations to other resources a new ontology has been developed: the Enzyme Mechanism Ontology, which has permitted the transfer of annotations to Mechanism, Annotation and Classification in Enzymes (MACiE) and UniProt Knowledge Base (UniProtKB) resources. The CSA database schema has been re-designed and both the CSA data and search capabilities are presented in a new modern web interface

The SWISS-MODEL Repository: new features and functionalities

The SWISS-MODEL Repository is a database of annotated 3D protein structure models generated by the SWISS-MODEL homology-modelling pipeline. As of September 2005, the repository contained 675 000 models for 604 000 different protein sequences of the UniProt database. Regular updates ensure that the content of the repository reflects the current state of sequence and structure databases, integrating new or modified target sequences, and making use of new template structures. Each Repository entry consists of one or more 3D models accompanied by detailed information about the target protein and the model building process: functional annotation, a detailed template selection log, target-template alignment, summary of the model building and model quality assessment. The SWISS-MODEL Repository is freely accessible at

Genetic heterogeneity in the leader and P1-coding regions of foot-and-mouth disease virus serotypes A and O in Africa

Genetic information regarding the leader (L) and complete capsid-coding (P1) region of FMD serotype A and O viruses prevalent on the African conti- nent is lacking.Here,we present the complete L-P1 sequences for eight serotype A and nine serotype O viruses recovered from FMDV outbreaks in East and West Africa over the last 33 years.Phylogenetic analysis of the P1 and capsid-coding regions revealed that the African isolates grouped according to serotype, and certain clusters were indicative of transboundary as well as intra-regional spread of the virus.However,similar analysis of the L region revealed random groupings of isolates from serotypes O and A.Comparisons between the phylogenetic trees derived from the structura lcoding regions and the L region pointed to a possibility of genetic recombination.The in- tertypic nucleotide and amino acid variation of all the isolates in this stud ysupported results from previous studies where the externally located 1D was the most variable whilst the internally located 1A was the most conserved,which likely reflects the selective pressures on these proteins.Amino acids identified previously as important for FMDV structure and functioning were found to be highly conserved.The information gained from this study will contribute to the construction of structurally designed FMDV vaccines in Africa.SA-UKcol-laboration initiative via the Department of Science and Technologyhttp://link.springer.com/journal/705hb201

Synthesis of empty African horse sickness virus particles

As a means to develop African horse sickness (AHS) vaccines that are safe and DIVA compliant, we investigated the synthesis of empty African horse sickness virus (AHSV) particles. The emphasis of this study was on the assembly of the major viral core (VP3 and VP7) and outer capsid proteins (VP2 and VP5) into architecturally complex, heteromultimeric nanosized particles. The production of fully assembled core-like particles (CLPs) was accomplished in vivo by baculovirus-mediated co-synthesis of VP3 and VP7. The two different outer capsid proteins were capable of associating independently of each other with preformed cores to yield partial virus-like particles (VLPs). Complete VLPs were synthesized, albeit with a low yield. Crystalline formation of AHSV VP7 trimers is thought to impede high-level CLP production. Consequently, we engineered and co-synthesized VP3 with a more hydrophilic mutant VP7, resulting in an increase in the turnover of CLPs.The Agricultural Research Council (ARC) and National Research Foundation (NRF).http://www.elsevier.com/locate/virusres2017-02-28hb2016GeneticsMicrobiology and Plant Patholog

Discovery: an interactive resource for the rational selection and comparison of putative drug target proteins in malaria

<p>Abstract</p> <p>Background</p> <p>Up to half a billion human clinical cases of malaria are reported each year, resulting in about 2.7 million deaths, most of which occur in sub-Saharan Africa. Due to the over-and misuse of anti-malarials, widespread resistance to all the known drugs is increasing at an alarming rate. Rational methods to select new drug target proteins and lead compounds are urgently needed. The Discovery system provides data mining functionality on extensive annotations of five malaria species together with the human and mosquito hosts, enabling the selection of new targets based on multiple protein and ligand properties.</p> <p>Methods</p> <p>A web-based system was developed where researchers are able to mine information on malaria proteins and predicted ligands, as well as perform comparisons to the human and mosquito host characteristics. Protein features used include: domains, motifs, EC numbers, GO terms, orthologs, protein-protein interactions, protein-ligand interactions and host-pathogen interactions among others. Searching by chemical structure is also available.</p> <p>Results</p> <p>An <it>in silico</it> system for the selection of putative drug targets and lead compounds is presented, together with an example study on the bifunctional DHFR-TS from <it>Plasmodium falciparum</it>.</p> <p>Conclusion</p> <p>The Discovery system allows for the identification of putative drug targets and lead compounds in Plasmodium species based on the filtering of protein and chemical properties.</p

Sequence-based prediction for vaccine strain selection and identification of antigenic variability in foot-and-mouth disease virus

Identifying when past exposure to an infectious disease will protect against newly emerging strains is central to understanding the spread and the severity of epidemics, but the prediction of viral cross-protection remains an important unsolved problem. For foot-and-mouth disease virus (FMDV) research in particular, improved methods for predicting this cross-protection are critical for predicting the severity of outbreaks within endemic settings where multiple serotypes and subtypes commonly co-circulate, as well as for deciding whether appropriate vaccine(s) exist and how much they could mitigate the effects of any outbreak. To identify antigenic relationships and their predictors, we used linear mixed effects models to account for variation in pairwise cross-neutralization titres using only viral sequences and structural data. We identified those substitutions in surface-exposed structural proteins that are correlates of loss of cross-reactivity. These allowed prediction of both the best vaccine match for any single virus and the breadth of coverage of new vaccine candidates from their capsid sequences as effectively as or better than serology. Sub-sequences chosen by the model-building process all contained sites that are known epitopes on other serotypes. Furthermore, for the SAT1 serotype, for which epitopes have never previously been identified, we provide strong evidence - by controlling for phylogenetic structure - for the presence of three epitopes across a panel of viruses and quantify the relative significance of some individual residues in determining cross-neutralization. Identifying and quantifying the importance of sites that predict viral strain cross-reactivity not just for single viruses but across entire serotypes can help in the design of vaccines with better targeting and broader coverage. These techniques can be generalized to any infectious agents where cross-reactivity assays have been carried out. As the parameterization uses pre-existing datasets, this approach quickly and cheaply increases both our understanding of antigenic relationships and our power to control disease

The SIB Swiss Institute of Bioinformatics' resources: focus on curated databases

The SIB Swiss Institute of Bioinformatics (www.isb-sib.ch) provides world-class bioinformatics databases, software tools, services and training to the international life science community in academia and industry. These solutions allow life scientists to turn the exponentially growing amount of data into knowledge. Here, we provide an overview of SIB's resources and competence areas, with a strong focus on curated databases and SIB's most popular and widely used resources. In particular, SIB's Bioinformatics resource portal ExPASy features over 150 resources, including UniProtKB/Swiss-Prot, ENZYME, PROSITE, neXtProt, STRING, UniCarbKB, SugarBindDB, SwissRegulon, EPD, arrayMap, Bgee, SWISS-MODEL Repository, OMA, OrthoDB and other databases, which are briefly described in this article

In silico structural characterisation of Plasmodium falciparum dihydro-6-hydroxymethylpterin pyrophosphokinase dihydropteroate synthase (PPPK-DHPS)

Malaria kills nearly 1.5 million and affects more than 500 million people annually, mostly in sub-Saharan Africa. The malaria parasite has developed resistance against almost all of the known drugs used for treatment. This fact has resulted in a constant battle between developing new anti-malarials and the parasite evolving resistance. One of the main drug combinations, pyrimethamine/sulfadoxine, targets the dihydrofolate reductase (DHFR) and dehydropteroate synthase (DHPS) proteins in the folate synthesis pathway of human malaria parasite, Plasmodium falciparum. The folate synthesis pathway is absent from the human host and thus presents itself as an ideal target for parasite-specific drugs. The three dimensional atomic coordinates of a target protein can help in designing new, more effective drugs. Malarial proteins are notoriously difficult to crystallize and thus homology modelling was chosen as an alternative method to obtain a protein structure. DHPS and PPPK occur as a bifunctional protein in the folate metabolism pathway. In this study, homology modelling was used to do in silico modelling of P. falciparum DHPS and hydroxymethyldihydropteridine pyrophosphokinase (PPPK). For the P. falciparum DHPS model the crystal structures of M. tuberculosis and B. anthracis DHPS were used as templates and for the P. falciparum PPPK model, the crystal structure ofE. coli PPPK. Molecular dynamics was used to investigate loop movement in DHPS and PPPK as well as to reveal the effect of resistance-causing mutations on sulfadoxine binding in P. falsiparum DHPS. This study revealed that four of the five known sulfadoxine resistance-causing mutations in DHPS disrupt the interaction between sulfadoxine and DHPS. This translates to a reduced capacity for sulfadoxine to inhibit DHPS, and results in resistance. The simulations also showed that both DHPS and PPPK have extensive loop movements during catalysis. The loop movements in DHPS and PPPK may also play a role in determining the catalytic rate of the enzymes. The work presented here provides researchers with models of P. falsiparum DHPS and PPPK. These models can be used to design experiments to investigate resistance, design new drugs and probe the structure of the PPPK-DHPS bifunctional enzyme.Dissertation (MSc (Biochemistry))--University of Pretoria, 2005.Biochemistryunrestricte