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Functional discoveries from a structural genomics approach to TB

Functional discoveries from a structural genomics approach to TB. Ted Baker. Palmerston North - centre of the Universe. Guy born and grew up here. Palmerston North. Oxford. York. Auckland. M. tuberculosis genome sequence. From the whole genome sequence: Mycobacterium tuberculosis.

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Functional discoveries from a structural genomics approach to TB

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  1. Functional discoveries from a structural genomics approach to TB Ted Baker

  2. Palmerston North - centre of the Universe Guy born and grew up here

  3. Palmerston North Oxford York Auckland

  4. M. tuberculosis genome sequence

  5. From the whole genome sequence: Mycobacterium tuberculosis • Approx. 3900 genes • Most functions assigned by homology (not direct experiment) • ~ 60% of unknown or uncertain function 15% found only in this organism • Many metabolic and other pathways appear incomplete or are not yet known at all • Can we use structure to discover function?

  6. Our programme at University of Auckland • Began ~ 1999 - pilot project focused on “unknown” proteins from Pyrobaculum aerophilum - a hyperthermophile • Switched in 2000 to larger project focused on Mycobacterium tuberculosis – cause of TB • As part of a larger worldwide network TB Structural Genomics Consortium - some central facilities in US, funded by NIH - primarily a focus for collaboration, coordination

  7. Target selection • P. aerophilum: - Proteins of unknown function, homologous with proteins from M. tuberculosis • M. tuberculosis: 1. Biosynthetic enzymes - potential drug targets 2. “Unknowns” important in TB biology eg genes upregulated in response to - hypoxia - antibiotics

  8. Progress to date • 120 ORFS cloned • 106 proteins expressed • 42 soluble • 25 crystallized • 15 structures solved(10 TB, 5 P. aerophilum) Bottlenecks- soluble expression - crystallization Not really high throughput

  9. 1. Biosynthetic enzymes LeuA - leucine MenB - menaquinone MshB - mycothiol MbtI - mycobactin TrpD - tryptophan MenG - menaquinone

  10. CH3 COO- CH3 CH3 C C CH2 COO- CH3 C C COO- H OH H O LeuA Nayden Koon, Chris Squire Catalyses first committed step in leucine biosynthesis Acetyl-CoA CoA α-isopropylmalate synthase α-ketoisovalerate α-isopropylmalate • Essential for growth of Mycobacterium tuberculosis - and for survival in lung (inside granulomas) • No equivalent enzyme in humans • Good potential drug target

  11. LeuA structure • Solved by MAD from SeMet protein – refined at 2.0 Å • In the first electron density maps we found: Bound a-KIV substrate molecule Zn ion – verified by fluorescence

  12. Overall structure Dimer 2 x 70 kDa N-terminal catalytic domain C-terminal domain 2 linker domains

  13. Function of C-terminal domain? • Activity lost when domain removed • Novel fold – duplicated bbba modules • Unusual juxtaposition of helix N-termini • Mutations that abolish feedback inhibition map here • Hypothesis: Site of leucine inhibition

  14. Leucine feedback inhibition Now shown crystallographically Hinge ?

  15. 2. “Unknowns” Rv2874 (DsbD) – EM domain Rv2238c – AhpE Rv1347c – acyltransferase Pa1218 = Rv0820 Pa2307 = Rv3735 Pa2754 = Rv1720c

  16. Pa_2754 and its homologues Vic Arcus, Kristina Backbro, Annette Roos • Small protein (16 kDa) of unknown function • Multiple homologues found in certain archaea P. aerophilum: Pa_0151, Pa_0285, Pa_0337, Pa_2754 • and in Mycobacteria M. tuberculosis: Rv0065, Rv0549, Rv0960, Rv1720c • Classified in Pfam database as PIN domains • More than 300 other examples… including > 30 examples in the M. tuberculosis genome • No fold prediction

  17. Structure determination – Pa_2754 • Soluble expression, crystallized easily BUT • Tetramer in solution, 2-3 tetramers in the crystal – no interpretable heavy atom derivative • Engineered 2 Met residues (L M mutations) • Structure solved by MAD phasing at 3.5 Å - found 15/16 Se (SOLVE) • Autobuilt and refined at 2.8 Å

  18. How to find function? 1. Multiple sequence alignment • Conserved Asp and Glu residues

  19. Conserved residues in Pa_2754 • Suggest conserved metal binding site Pa_2754 dimer

  20. How to find function? 2. Search of Protein Data Bank for proteins with similar fold • DALI search – a few very weak “hits”, none with Z > 3.0 - new fold? • Alexei Murzin similar to T4 phage RNase H - folds appear different, but central b-sheet and some helices are in common AND – conserved acidic residues match! • Hypothesis: protein is a nuclease • Proved by experiment! Cleaves single-stranded overhangs from double-stranded DNA • Common function for PIN domains?

  21. Why so many PIN domain proteins in M. tuberculosis? • Many PIN domains now discovered to be the toxin component in toxin:antitoxin (TA) pairs - occur in tandem (cassettes) on plasmids and bacterial chromosomes • Roles in plasmid maintenance, cell cycle arrest, and dormancy - Science (2003) 301, 1496-1499 • M. tuberculosis has at least 30 of these toxin:antitoxin pairs (all involving PIN domains) - role in dormancy? A T

  22. Acknowledgements Shaun Lott, Vic Arcus, Kristina Backbro, Heather Baker, Graeme Card, David Goldstone, Anthony Harrison, Jodie Johnston, Nayden Koon, Simon Li, Andrew McCarthy, Neil Peterson, Miriam Sharpe Marsden Fund of NZ Health Research Council of NZ New Economy Research Fund TB Structural Genomics Consortium Guy & Eleanor

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