Structural and Physiological Analyses of the Alkanesulphonate-Binding Protein (SsuA) of the Citrus Pathogen Xanthomonas citri.
PLoS One. 2013;8(11):e80083
Authors: Tófoli de Araújo F, Bolanos-Garcia VM, Pereira CT, Sanches M, Oshiro EE, Ferreira RC, Chigardze DY, Barbosa JA, de Souza Ferreira LC, Benedetti CE, Blundell TL, Balan A
BACKGROUND: The uptake of sulphur-containing compounds plays a pivotal role in the physiology of bacteria that live in aerobic soils where organosulfur compounds such as sulphonates and sulphate esters represent more than 95% of the available sulphur. Until now, no information has been available on the uptake of sulphonates by bacterial plant pathogens, particularly those of the Xanthomonas genus, which encompasses several pathogenic species. In the present study, we characterised the alkanesulphonate uptake system (Ssu) of Xanthomonas axonopodis pv. citri 306 strain (X. citri), the etiological agent of citrus canker.
METHODOLOGY/PRINCIPAL FINDINGS: A single operon-like gene cluster (ssuEDACB) that encodes both the sulphur uptake system and enzymes involved in desulphurisation was detected in the genomes of X. citri and of the closely related species. We characterised X. citri SsuA protein, a periplasmic alkanesulphonate-binding protein that, together with SsuC and SsuB, defines the alkanesulphonate uptake system. The crystal structure of SsuA bound to MOPS, MES and HEPES, which is herein described for the first time, provides evidence for the importance of a conserved dipole in sulphate group coordination, identifies specific amino acids interacting with the sulphate group and shows the presence of a rather large binding pocket that explains the rather wide range of molecules recognised by the protein. Isolation of an isogenic ssuA-knockout derivative of the X. citri 306 strain showed that disruption of alkanesulphonate uptake affects both xanthan gum production and generation of canker lesions in sweet orange leaves.
CONCLUSIONS/SIGNIFICANCE: The present study unravels unique structural and functional features of the X. citri SsuA protein and provides the first experimental evidence that an ABC uptake system affects the virulence of this phytopathogen.
PMID: 24282519 [PubMed - in process]
mCSM: predicting the effects of mutations in proteins using graph-based signatures.
Bioinformatics. 2013 Nov 26;
Authors: Pires DE, Ascher DB, Blundell TL
MOTIVATION: Mutations play fundamental roles in evolution by introducing diversity into genomes. Missense mutations in structural genes may become either selectively advantageous or disadvantageous to the organism by affecting protein stability and/or interfering with interactions between partners. Thus, the ability to predict the impact of mutations on protein stability and interactions is of significant value, particularly in understanding the effects of Mendelian and somatic mutations on the progression of disease. Here we propose a novel approach to the study of missense mutations, called mCSM, which relies on graph-based signatures. These encode distance patterns between atoms and are used to represent the protein residue environment and to train predictive models. In order to understand the roles of mutations in disease, we have evaluated their impacts not only on protein stability but also on protein-protein and protein-nucleic acid interactions.
RESULTS: We show that mCSM performs as well as or better than other methods that are used widely. mCSM signatures were successfully used in very different tasks demonstrating that the impact of a mutation can be correlated with the atomic-distance patterns surrounding an amino acid residue. We showed that mCSM can predict stability changes of a wide range of mutations occurring in the tumour suppressor protein p53, demonstrating the applicability of the proposed method in a challenging disease scenario.Availability and Implementation: A web server is available at http://structure.bioc.cam.ac.uk/mcsm CONTACT: email@example.com and firstname.lastname@example.org.
PMID: 24281696 [PubMed - as supplied by publisher]
Mycobacterium tuberculosis Dihydrofolate Reductase Reveals Two Conformational States and a Possible Low Affinity Mechanism to Antifolate Drugs.
Structure. 2013 Nov 6;
Authors: Dias MV, Tyrakis P, Domingues RR, Leme AF, Blundell TL
Inhibition of the biosynthesis of tetrahydrofolate (THF) has long been a focus in the treatment of both cancer and infectious diseases. Dihydrofolate reductase (DHFR), which catalyzes the last step, is one of the most thoroughly explored targets of this pathway, but there are no DHFR inhibitors used for tuberculosis treatment. Here, we report a structural, site-directed mutagenesis and calorimetric analysis of Mycobacterium tuberculosis DHFR (MtDHFR) in complex with classical DHFR inhibitors. Our study provides insights into the weak inhibition of MtDHFR by trimethoprim and other antifolate drugs, such as pyrimethamine and cycloguanil. The construction of the mutant Y100F, together with calorimetric studies, gives insights into low affinity of MtDHFR for classical DHFR inhibitors. Finally, the structures of MtDHFR in complex with pyrimethamine and cycloguanil define important interactions in the active site and provide clues to the more effective design of antibiotics targeted against MtDHFR.
PMID: 24210757 [PubMed - as supplied by publisher]
Integrated biophysical approach to fragment screening and validation for fragment-based lead discovery.
Proc Natl Acad Sci U S A. 2013 Aug 6;110(32):12984-9
Authors: Silvestre HL, Blundell TL, Abell C, Ciulli A
In fragment-based drug discovery, the weak affinities exhibited by fragments pose significant challenges for screening. Biophysical techniques are used to address this challenge, but there is no clear consensus on which cascade of methods is best suited to identify fragment hits that ultimately translate into bound X-ray structures and provide bona fide starting points for synthesis. We have benchmarked an integrated biophysical approach for fragment screening and validation against Mycobacterium tuberculosis pantothenate synthetase. A primary screen of 1,250 fragments library was performed by thermal shift, followed by secondary screen using one-dimensional NMR spectroscopy (water ligand observed gradient spectroscopy and saturation transfer difference binding experiments) and ultimate hit validation by isothermal titration calorimetry and X-ray crystallography. Our multibiophysical approach identified three distinct binding sites for fragments and laid a solid foundation for successful structure-based elaboration into potent inhibitors.
PMID: 23872845 [PubMed - indexed for MEDLINE]
Cooperative heparin-mediated oligomerization of fibroblast growth factor-1 (FGF1) precedes recruitment of FGFR2 to ternary complexes.
Biophys J. 2013 Apr 16;104(8):1720-30
Authors: Brown A, Robinson CJ, Gallagher JT, Blundell TL
Fibroblast growth factors (FGFs) utilize cell surface heparan sulfate as a coreceptor in the assembly of signaling complexes with FGF-receptors on the plasma membrane. Here we undertake a complete thermodynamic characterization of the assembly of the FGF signaling complex using isothermal titration calorimetry. Heparin fragments of defined length are used as chemical analogs of the sulfated domains of heparan sulfate and examined for their ability to oligomerize FGF1. Binding is modeled using the McGhee-von Hippel formalism for the cooperative binding of ligands to a monodimensional lattice. Oligomerization of FGFs on heparin is shown to be mediated by positive cooperativity (α = 6). Heparin octasaccharide is the shortest length capable of dimerizing FGF1 and on longer heparin chains FGF1 binds with a minimal footprint of 4.2 saccharide units. The thermodynamics and stoichiometry of the ternary complex suggest that in solution FGF1 binds to heparin in a trans-dimeric manner before FGFR recruitment.
PMID: 23601319 [PubMed - indexed for MEDLINE]
CREDO: a structural interactomics database for drug discovery.
Database (Oxford). 2013;2013:bat049
Authors: Schreyer AM, Blundell TL
CREDO is a unique relational database storing all pairwise atomic interactions of inter- as well as intra-molecular contacts between small molecules and macromolecules found in experimentally determined structures from the Protein Data Bank. These interactions are integrated with further chemical and biological data. The database implements useful data structures and algorithms such as cheminformatics routines to create a comprehensive analysis platform for drug discovery. The database can be accessed through a web-based interface, downloads of data sets and web services at http://www-cryst.bioc.cam.ac.uk/credo. Database URL: http://www-cryst.bioc.cam.ac.uk/credo.
PMID: 23868908 [PubMed - indexed for MEDLINE]
TIMBAL v2: update of a database holding small molecules modulating protein-protein interactions.
Database (Oxford). 2013;2013:bat039
Authors: Higueruelo AP, Jubb H, Blundell TL
TIMBAL is a database holding molecules of molecular weight <1200 Daltons that modulate protein-protein interactions. Since its first release, the database has been extended to cover 50 known protein-protein interactions drug targets, including protein complexes that can be stabilized by small molecules with therapeutic effect. The resource contains 14 890 data points for 6896 distinct small molecules. UniProt codes and Protein Data Bank entries are also included. Database URL: http://www-cryst.bioc.cam.ac.uk/timbal
PMID: 23766369 [PubMed - indexed for MEDLINE]
Structure of the catalytic region of DNA ligase IV in complex with an Artemis fragment sheds light on double-strand break repair.
Structure. 2013 Apr 2;21(4):672-9
Authors: Ochi T, Gu X, Blundell TL
Nonhomologous end joining (NHEJ) is central to the repair of double-stranded DNA breaks throughout the cell cycle and plays roles in the development of the immune system. Although three-dimensional structures of most components of NHEJ have been defined, those of the catalytic region of DNA ligase IV (LigIV), a specialized DNA ligase known to work in NHEJ, and of Artemis have remained unresolved. Here, we report the crystal structure at 2.4 Å resolution of the catalytic region of LigIV (residues 1-609) in complex with an Artemis peptide. We describe interactions of the DNA-binding domain of LigIV with the continuous epitope of Artemis, which, together, form a three-helix bundle. A kink in the first helix of LigIV introduced by a conserved VPF motif gives rise to a hydrophobic pocket, which accommodates a conserved tryptophan from Artemis. We provide structural insights into features of LigIV among human DNA ligases.
PMID: 23523427 [PubMed - indexed for MEDLINE]