A personal history of using crystals and crystallography to understand biology and advanced drug discovery

Over the past 60 years, the use of crystals to define structures of complexes using X-ray analysis has contributed to the discovery of new medicines in a very significant way. This has been in understanding not only small-molecule inhibitors of proteins, such as enzymes, but also protein or peptide hormones or growth factors that bind to cell surface receptors. Experimental structures from crystallography have also been exploited in software to allow prediction of structures of important targets based on knowledge of homologues. Crystals and crystallography continue to contribute to drug design and provide a successful example of academia–industry collaboration.</jats:p

Rappertk: a versatile engine for discrete restraint-based conformational sampling of macromolecules

Background: Macromolecular structures are modeled by conformational optimization within experimental and knowledge-based restraints. Discrete restraint-based sampling generates high-quality structures within these restraints and facilitates further refinement in a continuous all-atom energy landscape. This approach has been used successfully for protein loop modeling, comparative modeling and electron density fitting in X-ray crystallography.|Results: Here we present a software toolkit (Rappertk) which generalizes discrete restraint-based sampling for use in structural biology. Modular design and multi-layered architecture enables Rappertk to sample conformations of any macromolecule at many levels of detail and within a variety of experimental restraints. Performance against a C-alpha-tracing benchmark shows that the efficiency has not suffered despite the overhead required by this flexibility. We demonstrate the toolkit's capabilities by building high-quality beta-sheets and by introducing restraint-driven sampling. RNA sampling is demonstrated by rebuilding a protein-RNA interface. Ability to construct arbitrary ligands is used in sampling protein- ligand interfaces within electron density. Finally, secondary structure and shape information derived from EM are combined to generate multiple conformations of a protein consistent with the observed density.|Conclusion: Through its modular design and ease of use, Rappertk enables exploration of a wide variety of interesting avenues in structural biology. This toolkit, with illustrative examples, is freely available to academic users from http://www-cryst.bioc.cam.ac.uk/similar to swanand/mysite/rtk/index.html

Decoding the similarities and differences among mycobacterial species

Mycobacteriaceae comprises pathogenic species such as Mycobacterium tuberculosis, M. leprae and M. abscessus, as well as non-pathogenic species, for example, M. smegmatis and M. thermoresistibile. Genome comparison and annotation studies provide insights into genome evolutionary relatedness, identify unique and pathogenicity-related genes in each species, and explore new targets that could be used for developing new diagnostics and therapeutics. Here, we present a comparative analysis of ten-mycobacterial genomes with the objective of identifying similarities and differences between pathogenic and non-pathogenic species. We identified 1080 core orthologous clusters that were enriched in proteins involved in amino acid and purine/pyrimidine biosynthetic pathways, DNA-related processes (replication, transcription, recombination and repair), RNA-methylation and modification, and cell- wall polysaccharide biosynthetic pathways. For their pathogenicity and survival in the host cell, pathogenic species have gained specific sets of genes involved in repair and protection of their genomic DNA. M. leprae is of special interest owing to its having the smallest genome (1600 genes and ~1300 psuedogenes), yet poor genome annotation. More than 75% of the pseudogenes were found to have a functional ortholog in the other mycobacterial genomes and belong to protein families such as transferases, oxidoreductases and hydrolases.This work was supported by MRC Newton Award (RG78439: SM, TLB), Programme Grant (093167/Z/10/Z: TLB), Cystic Fibrosis Trust Grant (RG70975) and Wellcome Trust Investigator Award (200814/Z/16/Z: TLB), American Leprosy Mission (RG88726: SCV). Funding for open access charge: [MRC Newton Award/ RG78439]

Different DNA End Configurations Dictate Which NHEJ Components Are Most Important for Joining Efficiency

The nonhomologous DNA end-joining (NHEJ) pathway is a key mechanism for repairing dsDNA breaks that occur often in eukaryotic cells. In the simplest model, these breaks are first recognized by Ku, which then interacts with other NHEJ proteins to improve their affinity at DNA ends. These include DNA-PK$_{cs}$ and Artemis for trimming the DNA ends; DNA polymerase μ and λ to add nucleotides; and the DNA ligase IV complex to ligate the ends with the additional factors, XRCC4 (X-ray repair cross-complementing protein 4), XLF (XRCC4-like factor/Cernunos), and PAXX (paralog of XRCC4 and XLF). $\textit{In vivo}$ studies have demonstrated the degrees of importance of these NHEJ proteins in the mechanism of repair of dsDNA breaks, but interpretations can be confounded by other cellular processes. $\textit{In vitro}$ studies with NHEJ proteins have been performed to evaluate the nucleolytic resection, polymerization, and ligation steps, but a complete system has been elusive. Here we have developed a NHEJ reconstitution system that includes the nuclease, polymerase, and ligase components to evaluate relative NHEJ efficiency and analyze ligated junctional sequences for various types of DNA ends, including blunt, 5' overhangs, and 3' overhangs. We find that different dsDNA end structures have differential dependence on these enzymatic components. The dependence of some end joining on only Ku and XRCC4·DNA ligase IV allows us to formulate a physical model that incorporates nuclease and polymerase components as needed.National Institutes of Health, Cancer Research UK Program Grant IDs: C6/A11224, C6946/A14492), Wellcome Trust (Grant IDs: WT092096, WT093167

COSMIC Cancer Gene Census 3D database: understanding the impacts of mutations on cancer targets

Mutations in hallmark genes are believed to be the main drivers of cancer progression. These mutations are reported in the Catalogue of Somatic Mutations in Cancer (COSMIC). Structural appreciation of where these mutations appear, in protein-protein interfaces, active sites or deoxyribonucleic acid (DNA) interfaces, and predicting the impacts of these mutations using a variety of computational tools are crucial for successful drug discovery and development. Currently, there are 723 genes presented in the COSMIC Cancer Gene Census. Due to the complexity of the gene products, structures of only 87 genes have been solved experimentally with structural coverage between 90% and 100%. Here, we present a comprehensive, user-friendly, web interface (https://cancer-3d.com/) of 714 modelled cancer-related genes, including homo-oligomers, hetero-oligomers, transmembrane proteins and complexes with DNA, ribonucleic acid, ligands and co-factors. Using SDM and mCSM software, we have predicted the impacts of reported mutations on protein stability, protein-protein interfaces affinity and protein-nucleic acid complexes affinity. Furthermore, we also predicted intrinsically disordered regions using DISOPRED3

Achieving selectivity in space and time with DNA double-strand-break response and repair: molecular stages and scaffolds come with strings attached

When double-strand breaks (DSBs) in DNA remain unrepaired, catastrophic loss of genes occurs, leading to translocations, mutations and carcinogenesis. If a sister chromatid is not available at the DNA DSB, nonhomologous end joining (NHEJ) is used to join broken ends. The NHEJ pathway comprises synapsis, end processing and ligation. Here, we ask how DSBs in DNA are repaired efficiently. We suggest that colocation of proteins is achieved over time by the following components: stages, where the main actors are assembled, scaffolds that are erected quickly around broken parts to give access, and strings that tether proteins together. In NHEJ, a $\textit{stage}$ is provided by the Ku heterodimer interacting with DSBs and several other proteins including DNA-PKcs, APLF, BRCA1 and PAXX. A further $\textit{stage}$, DNA-PKcs, links the kinase with DNA, Ku, PARP1, BRCA1 and Artemis. A temporary $\textit{scaffold}$ facilitates repair and is constructed from XRCC4/XLF filaments that bridge Ku bound at DSB ends. LigIV bound to XRCC4 C-termini likely terminates the scaffold, bringing LigIV close to the DNA broken ends. A $\textit{string}$, provided by the Artemis C-terminal region, is intrinsically disordered but includes short linear ‘‘epitopes’’ that recognise DNA-PKcs, LigIV and PTIP, so keeping these components nearby. We show that these stages, scaffolds and strings facilitate colocation and efficient DSB repair. Understanding these processes provides insight into the biology of DNA repair and possible therapeutic intervention in cancer and other diseases.Wellcome Trust (Programme Grant ID: 093167/Z/ 10/Z), National Health and Medical Research Council of Australia (C. J. Martin Research Fellowship, Grant ID: APP1072476), Gates Cambridge ScholarshipThis is the final version of the article. It first appeared from Springer via http://dx.doi.org/10.1007/s11224-016-0841-

DNA-PKcs structure suggests an allosteric mechanism modulating DNA double-strand break repair

DNA-dependent protein kinase catalytic subunit (DNA-PKcs) is a central component of nonhomologous end joining (NHEJ), repairing DNA double-strand breaks that would otherwise lead to apoptosis or cancer. We have solved its structure in complex with the C-terminal peptide of Ku80 at 4.3 angstrom resolution using x-ray crystallography. We show that the 4128–amino acid structure comprises three large structural units: the N-terminal unit, the Circular Cradle, and the Head. Conformational differences between the two molecules in the asymmetric unit are correlated with changes in accessibility of the kinase active site, which are consistent with an allosteric mechanism to bring about kinase activation. The location of KU80ct194 in the vicinity of the breast cancer 1 (BRCA1) binding site suggests competition with BRCA1, leading to pathway selection between NHEJ and homologous recombination.This work was funded by the Wellcome Trust (grant 093167/Z/10/Z)

TIBLE: a web-based, freely accessible resource for small-molecule binding data for mycobacterial species

TIBLE is a web-based resource that provides easy access to data on the minimal inhibitory concentrations for small molecules against several mycobacterial species, as well as the target binding and off-target predictions for Mycobacterium tuberculosis. The current version of the database holds the activity data for more than 19 000 distinct small molecules against 39 mycobacterial species, binding data for 106 Mycobacterium tuberculosis target proteins and predictions for their potential off-targets. The resource integrates disparate public data and methods to provide easy access to the minimum inhibitory concentration and binding data, facilitation of data sharing, and identification of small molecules and targets for development of anti-tuberculosis therapeutics.Wellcome Trust Seeding Drug Discovery (101134/Z/13/Z to A.P.H., T.L.B.); MRC Newton Award (RG78439 to S.M. and T.L.B.); Programme Grant (093167/Z/10/Z to T.L.B.); Cystic Fibrosis Trust Grant (RG 70975); Wellcome Trust Investigator Award (200814/Z/16/Z to T.L.B.)

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/sdm2Gates HIT-TB and the EU MM4TB [Project ID: 260872 to A.P.P. and T.L.B.]; Bill & Melinda Gates Foundation [RG60453 to B.O.M.]; Jack Brockhoff Foundation [JBF 4186, 2016 to D.B.A.]; C.J. Martin Research Fellowship from the National Health and Medical Research Council of Australia [APP1072476]; Wellcome Trust Programme Grant [093167/Z/10/Z to D.B.A., T.L.B.]; Newton Fund RCUK-CONFAP Grant awarded by The Medical Research Council (MRC) [MR/M026302/1]. Funding for open access charge: Bill & Melinda Gates Foundation [RG60453] Gates HIT-TB; Wellcome Trust Programme Grant [093167/Z/10/Z]; Newton Fund RCUK-CONFAP Grant awarded by the Medical Research Council (MRC) [MR/M026302/1

Threonine 57 is required for the post-translational activation of Escherichia coli aspartate α-decarboxylase.

Aspartate α-decarboxylase is a pyruvoyl-dependent decarboxylase required for the production of β-alanine in the bacterial pantothenate (vitamin B5) biosynthesis pathway. The pyruvoyl group is formed via the intramolecular rearrangement of a serine residue to generate a backbone ester intermediate which is cleaved to generate an N-terminal pyruvoyl group. Site-directed mutagenesis of residues adjacent to the active site, including Tyr22, Thr57 and Tyr58, reveals that only mutation of Thr57 leads to changes in the degree of post-translational activation. The crystal structure of the site-directed mutant T57V is consistent with a non-rearranged backbone, supporting the hypothesis that Thr57 is required for the formation of the ester intermediate in activation