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Instruments / Technical Area	Publication Details	Published	Actions
I02-Macromolecular Crystallography I04-1-Macromolecular Crystallography (fixed wavelength)	Crystal structure of the TreS-Pep2 complex, initiating α-glucan synthesis in the GlgE pathway of mycobacteria Ali A. Kermani , Rana Roy , Chai Gopalasingam , Klaudia I. Kocurek , Trushar R. Patel , Luke J. Alderwick , Gurdyal S. Besra , Klaus Fütterer Journal Of Biological Chemistry DOI: 10.1074/jbc.RA118.004297 Diamond Proposal Number(s): [6388, 8359, 10369] Open Access Show Abstract ▼ Abstract: A growing body of evidence implicates the mycobacterial capsule - the outermost layer of the mycobacterial cell envelope - in modulation of the host immune response and virulence of mycobacteria. Mycobacteria synthesize the dominant capsule component, α(1→4)-linked glucan, via three interconnected, and potentially redundant metabolic pathways. Here, we report the crystal structure of the Mycobacterium smegmatis TreS-Pep2 complex, containing trehalose synthase (TreS) and maltokinase (Pep2), which convert trehalose to maltose-1-phosphate as part of the TreS-Pep2-GlgE pathway. The structure, at 3.6 Å resolution, revealed that a diamond-shaped TreS tetramer forms the core of the complex, and that pairs of Pep2 monomers bind to opposite apices of the tetramer in a 4+4 configuration. However, for the M. smegmatis orthologues, results from isothermal titration calorimetry and analytical ultracentrifugation experiments indicated that the prevalent stoichiometry in solution is 4 TreS + 2 Pep2 protomers. The observed discrepancy between the crystallised complex and the behaviour in the solution state may be explained by the relatively weak affinity of Pep2 for TreS (Kd 3.5 μM at mildly acidic pH) and crystal packing favouring the 4+4 complex. Proximity of the ATP binding site in Pep2 to the complex interface provides a rational basis for rate enhancement of Pep2 upon binding to TreS, but the complex structure appears to rule out substrate channeling between the active sites of TreS and Pep2. Our findings provide a structural model for the trehalose synthase-maltokinase complex in M. smegmatis that offers critical insights into capsule assembly.	Mar 2019
I04-Macromolecular Crystallography	Biochemical and structural characterization of mycobacterial aspartyl-tRNA synthetase AspS, a promising TB drug target Sudagar S. Gurcha , Veeraraghavan Usha , Jonathan A. G. Cox , Klaus Futterer , K Abrahams , Apoorva Bhatt , Luke Alderwick , Robert C. Reynolds , Nicholas J. Loman , Vijayashankar Nataraj , Carlos Alemparte , David Barros , Adrian J. Lloyd , Lluis Ballell , Judith V. Hobrath , Gurdyal S. Besra Plos One 9 (11) DOI: 10.1371/journal.pone.0113568 PMID: 25409504 Diamond Proposal Number(s): [10369] Open Access Show Abstract ▼ Abstract: The human pathogen Mycobacterium tuberculosis is the causative agent of pulmonary tuberculosis (TB), a disease with high worldwide mortality rates. Current treatment programs are under significant threat from multi-drug and extensively-drug resistant strains of M. tuberculosis, and it is essential to identify new inhibitors and their targets. We generated spontaneous resistant mutants in Mycobacterium bovis BCG in the presence of 10× the minimum inhibitory concentration (MIC) of compound 1, a previously identified potent inhibitor of mycobacterial growth in culture. Whole genome sequencing of two resistant mutants revealed in one case a single nucleotide polymorphism in the gene aspS at 535GAC>535AAC (D179N), while in the second mutant a single nucleotide polymorphism was identified upstream of the aspS promoter region. We probed whole cell target engagement by overexpressing either M. bovis BCG aspS or Mycobacterium smegmatis aspS, which resulted in a ten-fold and greater than ten-fold increase, respectively, of the MIC against compound 1. To analyse the impact of inhibitor 1 on M. tuberculosis AspS (Mt-AspS) activity we over-expressed, purified and characterised the kinetics of this enzyme using a robust tRNA-independent assay adapted to a high-throughput screening format. Finally, to aid hit-to-lead optimization, the crystal structure of apo M. smegmatis AspS was determined to a resolution of 2.4 Å.	Nov 2014

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