A recognition model of ACP-HCS interaction for programmed beta-branching in type I polyketide synthases
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Oral presentation at the Synthetic Biology of Antibiotic Production II (http://syntheticbio.esf.org), 30 August - 4 September 2014 at Sant Feliu de Guixols, Costa Brava, Spain.
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A recognition model of ACP-HCS interaction for programmed beta-branching in type I polyketide synthases
- 1. A recognition model of ACP-HCS interaction for programmed beta- branching in type I polyketide synthases Rohit Farmer
- 2. @rohitfarmer Haines et al., A conserved motif flags acyl carrier proteins for β-branching in polyketide synthesis. Nature Chemical Biology. 2013 Nov;9(11):685-92. #BranchOut
- 3. Outline @rohitfarmer • Introduction • Polyketides • Polyketide synthases (PKS) • Beta Branching • ACP-HCS interaction • Tryptophan defines branching ACPs • Modelling ACP-HCS interaction • Summary
- 4. Polyketide @rohitfarmer Polyketide • Natural compounds • Produced by bacteria, fungi and plants • Many have medicinal properties erythromycin A (antibiotic) rapamycin (immunosuppressant) doxorubicin (antitumor)
- 5. Polyketide synthases (PKS) @rohitfarmer Minimal Type I modular PKS Kapur S et al. PNAS 2012;109:4110-4115 ACP: Acyl carrier protein - holds substrate KS: Ketosynthase domain catalyzes chain elongation via decarboxylative condensation AT: AT domain catalyzes transfer of a selected extender unit
- 6. Mupirocin and Beta Branching @rohitfarmer Mupirocin ~ effective against MRSA Portion of mupirocin biosynthesis pathway; beta-branching mechanism Mupirocin
- 7. Questions?? @rohitfarmer How does the HCS cassette ‘know’ where to act? • Tandem array of ACPs? • Absence of KR? • Neighbouring sequence structure? • ACP structure? ACP : Acyl carrier protein - holds substrate KR : Ketoreductase - alters sidechain, incompatible with β-branch HCS cassette - introduces β-branch
- 8. Answers @rohitfarmer How does the HCS cassette ‘know’ where to act? • Tandem array of ACPs? • Absence of KR? • Neighbouring sequence structure? • ACP structure? ACP : Acyl carrier protein - holds substrate KR : Ketoreductase - alters sidechain, incompatible with β-branch HCS cassette - introduces β-branch
- 9. Tryptophan Defines the Branching ACPs @rohitfarmer Standard ACPs Branching ACPs From 15 well characterized PKS clusters known to have beta branching #TonyHaines
- 10. NMR solved ACP-A3a/b structures @rohitfarmer Mup-ACP-A3a/b di-domain structure #MattCrump Haines et al., Nat Chem Biol. 2013 Nov;9(11):685-92.
- 11. Residues 29-35 (central part of the loop between helix I and helix II), 41, 44 (in helix II), 57-59 (N-ter of helix III), and 64 (C-ter of helix 3) show significantly greater motion in the mutant than in the wild type. ACP-A3a/b MD simulation @rohitfarmer Haines et al., Nat Chem Biol. 2013 Nov;9(11):685-92.
- 12. Residues 29-35 (central part of the loop between helix I and helix II), 41, 44 (in helix II), 57-59 (N-ter of helix III), and 64 (C-ter of helix 3) show significantly greater motion in the mutant than in the wild type. ACP-A3a @rohitfarmer N C Helix I Helix II Loop I-II Helix III Helix IV
- 13. ACP + MupH Docking @rohitfarmer R HADDOCK MupHACP
- 14. ACP + MupH complex @rohitfarmer
- 15. ACP + MupH complex @rohitfarmer Orientation 1
- 16. ACP + MupH complex @rohitfarmer Orientation 2
- 17. ACP + MupH complex @rohitfarmer Dominant orientation (12 out of 16 complexes) Helix III
- 18. PIER and Evolutionary Trace Analysis @rohitfarmer PIER ET Haines et al., Nat Chem Biol. 2013 Nov;9(11):685-92.
- 19. ACP-A3a+MupH complex interface residues @rohitfarmer Haines et al., Nat Chem Biol. 2013 Nov;9(11):685-92.
- 20. ACP-A3a+MupH complex @rohitfarmer Haines et al., Nat Chem Biol. 2013 Nov;9(11):685-92. N C Trp44 Met419 Tyr62 Ser38 Polyketide intermediate ACP MupH Pin I Pin II
- 21. Y62 to F and A Mutation @rohitfarmer Haines et al., Nat Chem Biol. 2013 Nov;9(11):685-92.
- 22. ACP-A3a+MupH complex interface residues @rohitfarmer #ChrisMThomas Sequence alignment of MupH homologs 419
- 23. BatC L-M mutation @rohitfarmer Haines et al., Nat Chem Biol. 2013 Nov;9(11):685-92.
- 24. BatC L-M mutation @rohitfarmer Bioassay plates - IPTG + IPTG WT Δ mupH Δ mupH+ batC + batC batC WT batC L-M WT #EltonStephen Haines et al., Nat Chem Biol. 2013 Nov;9(11):685-92.
- 25. Kal ACP swap @rohitfarmer Bioassay plates WT ΔACP4 +K24a
- 26. Hidden Markov Models @rohitfarmer Training set for branching-ACPs Test set for branching-ACPs Virginiamycin cluster Leinamycin cluster Haines et al., Nat Chem Biol. 2013 Nov;9(11):685-92. Training set for Std-ACPs Test set for Std-ACPs
- 27. Hidden Markov Models @rohitfarmer Std ACP HMMsearch against TrEMBL and RefSeq microbial databases fetched 16,490 ACP like sequences. Unlisted variants in similar clusters Known branching Predicted branching ACPs that may add branches in a non-type I PKS pathway Insufficient sequence context or conflicting information Predicted nonbranching Sequences not examined Haines et al., Nat Chem Biol. 2013 Nov;9(11):685-92.
- 28. Summary @rohitfarmer Haines et al., Nat Chem Biol. 2013 Nov;9(11):685-92. #TonyHaines #BranchOut
- 29. Acknowledgement @rohitfarmer Supervisors Peter J. Winn Christopher M. Thomas Thomas Group Members Anthony Haines Yusra Alsammarraie Joanne Hothersall Elton R Stephens Claire Miller Rachel Gurney Yuiko Takebayashi Collaborators Matthew Crump, Bristol, UK Russell J Cox, Bristol, UK Christine L Willis, Bristol, UK Thomas J Simpson, Bristol, UK John Crosby, Bristol, UK Rob Lavigne, Heverlee, Belgium Special Thanks To Alexandre M.J.J. Bonvin PhD Scholarship The Darwin Trust of Edinburgh Facility BlueBEAR: UoB supercomputing facility