Synthetic viability genomic screening defines Sae2 function in DNA repair.

DNA double-strand break (DSB) repair by homologous recombination (HR) requires 3' single-stranded DNA (ssDNA) generation by 5' DNA-end resection. During meiosis, yeast Sae2 cooperates with the nuclease Mre11 to remove covalently bound Spo11 from DSB termini, allowing resection and HR to ensue. Mitotic roles of Sae2 and Mre11 nuclease have remained enigmatic, however, since cells lacking these display modest resection defects but marked DNA damage hypersensitivities. By combining classic genetic suppressor screening with high-throughput DNA sequencing, we identify Mre11 mutations that strongly suppress DNA damage sensitivities of sae2∆ cells. By assessing the impacts of these mutations at the cellular, biochemical and structural levels, we propose that, in addition to promoting resection, a crucial role for Sae2 and Mre11 nuclease activity in mitotic DSB repair is to facilitate the removal of Mre11 from ssDNA associated with DSB ends. Thus, without Sae2 or Mre11 nuclease activity, Mre11 bound to partly processed DSBs impairs strand invasion and HR.

XSuLT: a web server for structural annotation and representation of sequence-structure alignments.

The web server XSuLT, an enhanced version of the protein alignment annotation program JoY, formats a submitted multiple-sequence alignment using three-dimensional (3D) structural information in order to assist in the comparative analysis of protein evolution and in the optimization of alignments for comparative modelling and construct design. In addition to the features analysed by JoY, which include secondary structure, solvent accessibility and sidechain hydrogen bonds, XSuLT annotates each amino acid residue with residue depth, chain and ligand interactions, inter-residue contacts, sequence entropy, root mean square deviation and secondary structure and disorder prediction. It is also now integrated with built-in 3D visualization which interacts with the formatted alignment to facilitate inspection and understanding. Results can be downloaded as stand-alone HTML for the formatted alignment and as XML with the underlying annotation data. XSuLT is freely available at http://structure.bioc.cam.ac.uk/xsult/.

SDM: a server for predicting effects of mutations on protein stability.

Here, we report a webserver for the improved SDM, used for predicting the effects of mutations on protein stability. As a pioneering knowledge-based approach, SDM has been highlighted as the most appropriate method to use in combination with many other approaches. We have updated the environment-specific amino-acid substitution tables based on the current expanded PDB (a 5-fold increase in information), and introduced new residue-conformation and interaction parameters, including packing density and residue depth. The updated server has been extensively tested using a benchmark containing 2690 point mutations from 132 different protein structures. The revised method correlates well against the hypothetical reverse mutations, better than comparable methods built using machine-learning approaches, highlighting the strength of our knowledge-based approach for identifying stabilising mutations. Given a PDB file (a Protein Data Bank file format containing the 3D coordinates of the protein atoms), and a point mutation, the server calculates the stability difference score between the wildtype and mutant protein. The server is available at http://structure.bioc.cam.ac.uk/sdm2.

Genome3D: exploiting structure to help users understand their sequences.

Genome3D (http://www.genome3d.eu) is a collaborative resource that provides predicted domain annotations and structural models for key sequences. Since introducing Genome3D in a previous NAR paper, we have substantially extended and improved the resource. We have annotated representatives from Pfam families to improve coverage of diverse sequences and added a fast sequence search to the website to allow users to find Genome3D-annotated sequences similar to their own. We have improved and extended the Genome3D data, enlarging the source data set from three model organisms to 10, and adding VIVACE, a resource new to Genome3D. We have analysed and updated Genome3D's SCOP/CATH mapping. Finally, we have improved the superposition tools, which now give users a more powerful interface for investigating similarities and differences between structural models.

Structure-guided, target-based drug discovery - exploiting genome information from HIV to mycobacterial infections.

The use of protein crystallography in structure-guided drug discovery allows identification of potential inhibitor-binding sites and optimisation of interactions of hits and lead compounds with a target protein. An early example of this approach was the use of the structure of HIV protease in designing AIDS antivirals. More recently, use of structure-guided design with fragment-based drug discovery, which reduces the size of screening libraries by decreasing complexity, has improved ligand efficiency in drug design. Here, we discuss the use of structure-guided target identification and lead optimisation using fragment-based approaches in the development of new antimicrobials for mycobacterial infections.

Genome3D: a UK collaborative project to annotate genomic sequences with predicted 3D structures based on SCOP and CATH domains.

Genome3D, available at http://www.genome3d.eu, is a new collaborative project that integrates UK-based structural resources to provide a unique perspective on sequence-structure-function relationships. Leading structure prediction resources (DomSerf, FUGUE, Gene3D, pDomTHREADER, Phyre and SUPERFAMILY) provide annotations for UniProt sequences to indicate the locations of structural domains (structural annotations) and their 3D structures (structural models). Structural annotations and 3D model predictions are currently available for three model genomes (Homo sapiens, E. coli and baker's yeast), and the project will extend to other genomes in the near future. As these resources exploit different strategies for predicting structures, the main aim of Genome3D is to enable comparisons between all the resources so that biologists can see where predictions agree and are therefore more trusted. Furthermore, as these methods differ in whether they build their predictions using CATH or SCOP, Genome3D also contains the first official mapping between these two databases. This has identified pairs of similar superfamilies from the two resources at various degrees of consensus (532 bronze pairs, 527 silver pairs and 370 gold pairs).

Respiratory flexibility in response to inhibition of cytochrome C oxidase in Mycobacterium tuberculosis.

We report here a series of five chemically diverse scaffolds that have in vitro activities on replicating and hypoxic nonreplicating bacilli by targeting the respiratory bc1 complex in Mycobacterium tuberculosis in a strain-dependent manner. Deletion of the cytochrome bd oxidase generated a hypersusceptible mutant in which resistance was acquired by a mutation in qcrB. These results highlight the promiscuity of the bc1 complex and the risk of targeting energy metabolism with new drugs.

A platform for target prediction of phenotypic screening hit molecules.

Many drug discovery programmes, particularly for infectious diseases, are conducted phenotypically. Identifying the targets of phenotypic screening hits experimentally can be complex, time-consuming, and expensive. However, it would be valuable to know what the molecular target(s) is, as knowledge of the binding pose of the hit molecule in the binding site can facilitate the compound optimisation. Furthermore, knowing the target would allow de-prioritisation of less attractive chemical series or molecular targets. To generate target-hypotheses for phenotypic active compounds, an in silico platform was developed that utilises both ligand and protein-structure information to generate a ranked set of predicted molecular targets. As a result of the web-based workflow the user obtains a set of 3D structures of the predicted targets with the active molecule bound. The platform was exemplified using Mycobacterium tuberculosis, the causative organism of tuberculosis. In a test that we performed, the platform was able to predict the targets of 60% of compounds investigated, where there was some similarity to a ligand in the protein database.

'Candidatus Ornithobacterium hominis': insights gained from draft genomes obtained from nasopharyngeal swabs.

'Candidatus Ornithobacterium hominis' represents a new member of the Flavobacteriaceae detected in 16S rRNA gene surveys of people from South-East Asia, Africa and Australia. It frequently colonizes the infant nasopharynx at high proportional abundance, and we demonstrate its presence in 42 % of nasopharyngeal swabs from 12-month-old children in the Maela refugee camp in Thailand. The species, a Gram-negative bacillus, has not yet been cultured, but the cells can be identified in mixed samples by fluorescent hybridization. Here, we report seven genomes assembled from metagenomic data, two to improved draft standard. The genomes are approximately 1.9 Mb, sharing 62 % average amino acid identity with the only other member of the genus, the bird pathogen Ornithobacterium rhinotracheale. The draft genomes encode multiple antibiotic-resistance genes, competition factors, Flavobacterium johnsoniae-like gliding motility genes and a homologue of the Pasteurella multocida mitogenic toxin. Intra- and inter-host genome comparison suggests that colonization with this bacterium is both persistent and strain exclusive.

Arpeggio: A Web Server for Calculating and Visualising Interatomic Interactions in Protein Structures.

Interactions between proteins and their ligands, such as small molecules, other proteins, and DNA, depend on specific interatomic interactions that can be classified on the basis of atom type and distance and angle constraints. Visualisation of these interactions provides insights into the nature of molecular recognition events and has practical uses in guiding drug design and understanding the structural and functional impacts of mutations. We present Arpeggio, a web server for calculating interactions within and between proteins and protein, DNA, or small-molecule ligands, including van der Waals', ionic, carbonyl, metal, hydrophobic, and halogen bond contacts, and hydrogen bonds and specific atom-aromatic ring (cation-π, donor-π, halogen-π, and carbon-π) and aromatic ring-aromatic ring (π-π) interactions, within user-submitted macromolecule structures. PyMOL session files can be downloaded, allowing high-quality publication images of the interactions to be generated. Arpeggio is implemented in Python and available as a user-friendly web interface at http://structure.bioc.cam.ac.uk/arpeggio/ and as a downloadable package at https://bitbucket.org/harryjubb/arpeggio.

Mutations at protein-protein interfaces: Small changes over big surfaces have large impacts on human health.

Many essential biological processes including cell regulation and signalling are mediated through the assembly of protein complexes. Changes to protein-protein interaction (PPI) interfaces can affect the formation of multiprotein complexes, and consequently lead to disruptions in interconnected networks of PPIs within and between cells, further leading to phenotypic changes as functional interactions are created or disrupted. Mutations altering PPIs have been linked to the development of genetic diseases including cancer and rare Mendelian diseases, and to the development of drug resistance. The importance of these protein mutations has led to the development of many resources for understanding and predicting their effects. We propose that a better understanding of how these mutations affect the structure, function, and formation of multiprotein complexes provides novel opportunities for tackling them, including the development of small-molecule drugs targeted specifically to mutated PPIs.

Structural Biology and the Design of New Therapeutics: From HIV and Cancer to Mycobacterial Infections: A Paper Dedicated to John Kendrew.

Interest in applications of protein crystallography to medicine was evident, as the first high-resolution structures emerged in the 50s and 60s. In Cambridge, Max Perutz and John Kendrew sought to understand mutations in sickle cell and other genetic diseases related to hemoglobin, while in Oxford, the group of Dorothy Hodgkin became interested in long-lasting zinc-insulin crystals for treatment of diabetes and later considered insulin redesign, as synthetic insulins became possible. The use of protein crystallography in structure-guided drug discovery emerged as enzyme structures allowed the identification of potential inhibitor-binding sites and optimization of interactions of hits using the structure of the target protein. Early examples of this approach were the use of the structure of renin to design antihypertensives and the structure of HIV protease in design of AIDS antivirals. More recently, use of structure-guided design with fragment-based drug discovery, which reduces the size of screening libraries by decreasing complexity, has improved ligand efficiency in drug design and has been used to progress three oncology drugs through clinical trials to FDA approval. We exemplify current developments in structure-guided target identification and fragment-based lead discovery with efforts to develop new antimicrobials for mycobacterial infections.

CHOPIN: a web resource for the structural and functional proteome of Mycobacterium tuberculosis.

Tuberculosis kills more than a million people annually and presents increasingly high levels of resistance against current first line drugs. Structural information about Mycobacterium tuberculosis (Mtb) proteins is a valuable asset for the development of novel drugs and for understanding the biology of the bacterium; however, only about 10% of the ∼4000 proteins have had their structures determined experimentally. The CHOPIN database assigns structural domains and generates homology models for 2911 sequences, corresponding to ∼73% of the proteome. A sophisticated pipeline allows multiple models to be created using conformational states characteristic of different oligomeric states and ligand binding, such that the models reflect various functional states of the proteins. Additionally, CHOPIN includes structural analyses of mutations potentially associated with drug resistance. Results are made available at the web interface, which also serves as an automatically updated repository of all published Mtb experimental structures. Its RESTful interface allows direct and flexible access to structures and metadata via intuitive URLs, enabling easy programmatic use of the models.

Enriching the annotation of Mycobacterium tuberculosis H37Rv proteome using remote homology detection approaches: insights into structure and function.

The availability of the genome sequence of Mycobacterium tuberculosis H37Rv has encouraged determination of large numbers of protein structures and detailed definition of the biological information encoded therein; yet, the functions of many proteins in M. tuberculosis remain unknown. The emergence of multidrug resistant strains makes it a priority to exploit recent advances in homology recognition and structure prediction to re-analyse its gene products. Here we report the structural and functional characterization of gene products encoded in the M. tuberculosis genome, with the help of sensitive profile-based remote homology search and fold recognition algorithms resulting in an enhanced annotation of the proteome where 95% of the M. tuberculosis proteins were identified wholly or partly with information on structure or function. New information includes association of 244 proteins with 205 domain families and a separate set of new association of folds to 64 proteins. Extending structural information across uncharacterized protein families represented in the M. tuberculosis proteome, by determining superfamily relationships between families of known and unknown structures, has contributed to an enhancement in the knowledge of structural content. In retrospect, such superfamily relationships have facilitated recognition of probable structure and/or function for several uncharacterized protein families, eventually aiding recognition of probable functions for homologous proteins corresponding to such families. Gene products unique to mycobacteria for which no functions could be identified are 183. Of these 18 were determined to be M. tuberculosis specific. Such pathogen-specific proteins are speculated to harbour virulence factors required for pathogenesis. A re-annotated proteome of M. tuberculosis, with greater completeness of annotated proteins and domain assigned regions, provides a valuable basis for experimental endeavours designed to obtain a better understanding of pathogenesis and to accelerate the process of drug target discovery.

SInCRe-structural interactome computational resource for Mycobacterium tuberculosis.

We have developed an integrated database for Mycobacterium tuberculosis H37Rv (Mtb) that collates information on protein sequences, domain assignments, functional annotation and 3D structural information along with protein-protein and protein-small molecule interactions. SInCRe (Structural Interactome Computational Resource) is developed out of CamBan (Cambridge and Bangalore) collaboration. The motivation for development of this database is to provide an integrated platform to allow easily access and interpretation of data and results obtained by all the groups in CamBan in the field of Mtb informatics. In-house algorithms and databases developed independently by various academic groups in CamBan are used to generate Mtb-specific datasets and are integrated in this database to provide a structural dimension to studies on tuberculosis. The SInCRe database readily provides information on identification of functional domains, genome-scale modelling of structures of Mtb proteins and characterization of the small-molecule binding sites within Mtb. The resource also provides structure-based function annotation, information on small-molecule binders including FDA (Food and Drug Administration)-approved drugs, protein-protein interactions (PPIs) and natural compounds that bind to pathogen proteins potentially and result in weakening or elimination of host-pathogen protein-protein interactions. Together they provide prerequisites for identification of off-target binding.

Crystal structure of folliculin reveals a hidDENN function in genetically inherited renal cancer.

Mutations in the renal tumour suppressor protein, folliculin, lead to proliferative skin lesions, lung complications and renal cell carcinoma. Folliculin has been reported to interact with AMP-activated kinase, a key component of the mammalian target of rapamycin pathway. Most cancer-causing mutations lead to a carboxy-terminal truncation of folliculin, pointing to a functional importance of this domain in tumour suppression. We present here the crystal structure of folliculin carboxy-terminal domain and demonstrate that it is distantly related to differentially expressed in normal cells and neoplasia (DENN) domain proteins, a family of Rab guanine nucleotide exchange factors (GEFs). Using biochemical analysis, we show that folliculin has GEF activity, indicating that folliculin is probably a distantly related member of this class of Rab GEFs.