Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Mechanism of intersubunit ketosynthase–dehydratase interaction in polyketide synthases

Nature Chemical Biology volume 14, pages 270–275 (2018)Cite this article

Subjects

Abstract

Modular polyketide synthases (PKSs) produce numerous structurally complex natural products that have diverse applications in medicine and agriculture. PKSs typically consist of several multienzyme subunits that utilize structurally defined docking domains (DDs) at their N and C termini to ensure correct assembly into functional multiprotein complexes. Here we report a fundamentally different mechanism for subunit assembly in trans-acyltransferase (trans-AT) modular PKSs at the junction between ketosynthase (KS) and dehydratase (DH) domains. This mechanism involves direct interaction of a largely unstructured docking domain (DD) at the C terminus of the KS with the surface of the downstream DH. Acyl transfer assays and mechanism-based crosslinking established that the DD is required for the KS to communicate with the acyl carrier protein appended to the DH. Two distinct regions for binding of the DD to the DH were identified using NMR spectroscopy, carbene footprinting, and mutagenesis, providing a foundation for future elucidation of the molecular basis for interaction specificity.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Bioinformatics analysis of DHD domains and examples from the gladiolin and bacillaene PKSs.
Figure 2: Acyl transfer and protein crosslinking assays demonstrate that DHD domains play a key role in communication across KS/DH interfaces.
Figure 3: NMR titrations reveal two regions of DHD domains involved in the interaction with DH-ACP di-domains.
Figure 4: Mapping DHD and ACP domain interaction sites on the DH domain.
Figure 5: DHD domains interact selectively with their cognate DH domain partners.

Similar content being viewed by others

Accession codes

Primary accessions

Biological Magnetic Resonance Data Bank

Referenced accessions

Protein Data Bank

References

  1. Hertweck, C. The biosynthetic logic of polyketide diversity. Angew. Chem. Int. Ed. Engl. 48, 4688–4716 (2009).

    Article  CAS  PubMed  Google Scholar 

  2. Keatinge-Clay, A.T. The structures of type I polyketide synthases. Nat. Prod. Rep. 29, 1050–1073 (2012).

    Article  CAS  PubMed  Google Scholar 

  3. Richter, C.D., Nietlispach, D., Broadhurst, R.W. & Weissman, K.J. Multienzyme docking in hybrid megasynthetases. Nat. Chem. Biol. 4, 75–81 (2008).

    Article  CAS  PubMed  Google Scholar 

  4. Weissman, K.J. & Müller, R. Protein-protein interactions in multienzyme megasynthetases. ChemBioChem 9, 826–848 (2008).

    Article  CAS  PubMed  Google Scholar 

  5. Fischbach, M.A. & Walsh, C.T. Assembly-line enzymology for polyketide and nonribosomal peptide antibiotics: logic, machinery, and mechanisms. Chem. Rev. 106, 3468–3496 (2006).

    Article  CAS  PubMed  Google Scholar 

  6. Keatinge-Clay, A.T. Stereocontrol within polyketide assembly lines. Nat. Prod. Rep. 33, 141–149 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Wagner, D.T. et al. α-Methylation follows condensation in the gephyronic acid modular polyketide synthase. Chem. Commun. (Camb.) 52, 8822–8825 (2016).

    Article  CAS  Google Scholar 

  8. Helfrich, E.J.N. & Piel, J. Biosynthesis of polyketides by trans-AT polyketide synthases. Nat. Prod. Rep. 33, 231–316 (2016).

    Article  CAS  PubMed  Google Scholar 

  9. Katz, L. The DEBS paradigm for type I modular polyketide synthases and beyond. Methods Enzymol. 459, 113–142 (2009).

    Article  CAS  PubMed  Google Scholar 

  10. Broadhurst, R.W., Nietlispach, D., Wheatcroft, M.P., Leadlay, P.F. & Weissman, K.J. The structure of docking domains in modular polyketide synthases. Chem. Biol. 10, 723–731 (2003).

    Article  CAS  PubMed  Google Scholar 

  11. Gokhale, R.S. & Khosla, C. Role of linkers in communication between protein modules. Curr. Opin. Chem. Biol. 4, 22–27 (2000).

    Article  CAS  PubMed  Google Scholar 

  12. Buchholz, T.J. et al. Structural basis for binding specificity between subclasses of modular polyketide synthase docking domains. ACS Chem. Biol. 4, 41–52 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Kuo, J., Lynch, S.R., Liu, C.W., Xiao, X. & Khosla, C. Partial in vitro reconstitution of an orphan polyketide synthase associated with clinical cases of nocardiosis. ACS Chem. Biol. 11, 2636–2641 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Gulder, T., Freeman, M. & Piel, J. The catalytic diversity of multimodular polyketide synthases: natural product biosynthesis beyond textbook assembly rules. Top. Curr. Chem. https://doi.org/10.1007/128_2010_113 (2011).

  15. Dorival, J. et al. Characterization of intersubunit communication in the virginiamycin trans-acyl transferase polyketide synthase. J. Am. Chem. Soc. 138, 4155–4167 (2016).

    Article  CAS  PubMed  Google Scholar 

  16. Zeng, J. et al. Portability and structure of the four-helix bundle docking domains of trans-acyltransferase modular polyketide synthases. ACS Chem. Biol. 11, 2466–2474 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Song, L. et al. Discovery and biosynthesis of gladiolin: a Burkholderia gladioli antibiotic with promising activity against Mycobacterium tuberculosis. J. Am. Chem. Soc. 139, 7974–7981 (2017).

    Article  CAS  PubMed  Google Scholar 

  18. Eddy, S.R. Profile hidden Markov models. Bioinformatics 14, 755–763 (1998).

    Article  CAS  PubMed  Google Scholar 

  19. McGuffin, L.J., Bryson, K. & Jones, D.T. The PSIPRED protein structure prediction server. Bioinformatics 16, 404–405 (2000).

    Article  CAS  PubMed  Google Scholar 

  20. Dosztányi, Z., Csizmok, V., Tompa, P. & Simon, I. IUPred: web server for the prediction of intrinsically unstructured regions of proteins based on estimated energy content. Bioinformatics 21, 3433–3434 (2005).

    Article  PubMed  Google Scholar 

  21. Dosztányi, Z., Mészáros, B. & Simon, I. ANCHOR: web server for predicting protein binding regions in disordered proteins. Bioinformatics 25, 2745–2746 (2009).

    Article  PubMed  PubMed Central  Google Scholar 

  22. Worthington, A.S., Rivera, H., Torpey, J.W., Alexander, M.D. & Burkart, M.D. Mechanism-based protein cross-linking probes to investigate carrier protein-mediated biosynthesis. ACS Chem. Biol. 1, 687–691 (2006).

    Article  CAS  PubMed  Google Scholar 

  23. Kapur, S. et al. Mechanism based protein crosslinking of domains from the 6-deoxyerythronolide B synthase. Bioorg. Med. Chem. Lett. 18, 3034–3038 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Kosol, S., Contreras-Martos, S., Cedeño, C. & Tompa, P. Structural characterization of intrinsically disordered proteins by NMR spectroscopy. Molecules 18, 10802–10828 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Jensen, M.R., Ruigrok, R.W. & Blackledge, M. Describing intrinsically disordered proteins at atomic resolution by NMR. Curr. Opin. Struct. Biol. 23, 426–435 (2013).

    Article  CAS  PubMed  Google Scholar 

  26. Uversky, V.N. Functional roles of transiently and intrinsically disordered regions within proteins. FEBS J. 282, 1182–1189 (2015).

    Article  CAS  PubMed  Google Scholar 

  27. Manzi, L. et al. Carbene footprinting accurately maps binding sites in protein-ligand and protein-protein interactions. Nat. Commun. 7, 13288 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Pavlović-Lažetić, G.M. et al. Bioinformatics analysis of disordered proteins in prokaryotes. BMC Bioinformatics 12, 66 (2011).

    Article  PubMed  PubMed Central  Google Scholar 

  29. Vucetic, S. et al. DisProt: a database of protein disorder. Bioinformatics 21, 137–140 (2005).

    Article  CAS  PubMed  Google Scholar 

  30. Wright, P.E. & Dyson, H.J. Intrinsically disordered proteins in cellular signalling and regulation. Nat. Rev. Mol. Cell Biol. 16, 18–29 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Lin, Y.S., Hsu, W.L., Hwang, J.K. & Li, W.H. Proportion of solvent-exposed amino acids in a protein and rate of protein evolution. Mol. Biol. Evol. 24, 1005–1011 (2007).

    Article  CAS  PubMed  Google Scholar 

  32. Nilsson, J., Grahn, M. & Wright, A.P.H. Proteome-wide evidence for enhanced positive Darwinian selection within intrinsically disordered regions in proteins. Genome Biol. 12, R65 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Bellay, J. et al. Bringing order to protein disorder through comparative genomics and genetic interactions. Genome Biol. 12, R14 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Neduva, V. & Russell, R.B. Peptides mediating interaction networks: new leads at last. Curr. Opin. Biotechnol. 17, 465–471 (2006).

    Article  CAS  PubMed  Google Scholar 

  35. Gitlin, L., Hagai, T., LaBarbera, A., Solovey, M. & Andino, R. Rapid evolution of virus sequences in intrinsically disordered protein regions. PLoS Pathog. 10, e1004529 (2014).

    Article  PubMed  PubMed Central  Google Scholar 

  36. Keatinge-Clay, A. Crystal structure of the erythromycin polyketide synthase dehydratase. J. Mol. Biol. 384, 941–953 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  37. Wagner, D.T. et al. Structural and functional trends in dehydrating bimodules from trans-acyltransferase polyketide synthases. Structure 25, 1045–1055 e2 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Larkin, M.A. et al. Clustal W and Clustal X version 2.0. Bioinformatics 23, 2947–2948 (2007).

    Article  CAS  PubMed  Google Scholar 

  39. Worthington, A.S. & Burkart, M.D. One-pot chemo-enzymatic synthesis of reporter-modified proteins. Org. Biomol. Chem. 4, 44–46 (2006).

    Article  CAS  PubMed  Google Scholar 

  40. Meier, J.L., Mercer, A.C., Rivera, H. Jr. & Burkart, M.D. Synthesis and evaluation of bioorthogonal pantetheine analogues for in vivo protein modification. J. Am. Chem. Soc. 128, 12174–12184 (2006).

    Article  CAS  PubMed  Google Scholar 

  41. Vranken, W.F. et al. The CCPN data model for NMR spectroscopy: development of a software pipeline. Proteins 59, 687–696 (2005).

    Article  CAS  PubMed  Google Scholar 

  42. Lescop, E., Schanda, P. & Brutscher, B. A set of BEST triple-resonance experiments for time-optimized protein resonance assignment. J. Magn. Reson. 187, 163–169 (2007).

    Article  CAS  PubMed  Google Scholar 

  43. Palmer, A.G., III. NMR characterization of the dynamics of biomacromolecules. Chem. Rev. 104, 3623–3640 (2004).

    Article  CAS  PubMed  Google Scholar 

  44. Williamson, M.P. Using chemical shift perturbation to characterise ligand binding. Prog. Nucl. Magn. Reson. Spectrosc. 73, 1–16 (2013).

    Article  CAS  PubMed  Google Scholar 

  45. Schumann, F.H. et al. Combined chemical shift changes and amino acid specific chemical shift mapping of protein-protein interactions. J. Biomol. NMR 39, 275–289 (2007).

    Article  CAS  PubMed  Google Scholar 

  46. Whitmore, L. & Wallace, B.A. DICHROWEB, an online server for protein secondary structure analyses from circular dichroism spectroscopic data. Nucleic Acids Res. 32, W668–W673 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Lees, J.G., Miles, A.J., Wien, F. & Wallace, B.A. A reference database for circular dichroism spectroscopy covering fold and secondary structure space. Bioinformatics 22, 1955–1962 (2006).

    Article  CAS  PubMed  Google Scholar 

  48. Yang, J. et al. The I-TASSER Suite: protein structure and function prediction. Nat. Methods 12, 7–8 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Chen, V.B. et al. MolProbity: all-atom structure validation for macromolecular crystallography. Acta Crystallogr. D Biol. Crystallogr. 66, 12–21 (2010).

    CAS  PubMed  Google Scholar 

  50. Pierce, B.G. et al. ZDOCK server: interactive docking prediction of protein-protein complexes and symmetric multimers. Bioinformatics 30, 1771–1773 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

This research was supported by grants from the BBSRC (BB/L021692/1 to G.L.C. and BB/L022761/1 to J.R.L.). The Bruker MaXis II instrument used in this study was funded by the BBSRC (BB/M017982/1). N.J.O., J.E.M., L.M. and A.S.B. thank the University of Nottingham for funding. The research leading to these results has received funding from the European Research Council under the European Union's Seventh Framework Programme (FP/2007-2013)/ERC Grant Agreement 639907 (to J.R.L.). G.L.C. is the recipient of a Wolfson Research Merit Award from the Royal Society (WM130033). We thank L. Song for assistance with LC-MS analyses.

Author information

Authors and Affiliations

  1. Department of Chemistry, University of Warwick, Coventry CV4 7AL, UK

    Matthew Jenner, Simone Kosol, Daniel Griffiths, Panward Prasongpholchai, Józef R Lewandowski & Gregory L Challis

  2. Warwick Integrative Synthetic Biology Centre, University of Warwick, Coventry CV4 7AL, UK

    Matthew Jenner & Gregory L Challis

  3. School of Chemistry, University of Nottingham, Nottingham NG7 2RD, UK

    Lucio Manzi, Andrew S Barrow, John E Moses & Neil J Oldham

Authors
  1. Matthew Jenner
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Simone Kosol
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Daniel Griffiths
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Panward Prasongpholchai
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Lucio Manzi
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Andrew S Barrow
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. John E Moses
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Neil J Oldham
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Józef R Lewandowski
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Gregory L Challis
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

M.J. and G.L.C. designed the study, and all authors contributed to performing the research as follows: M.J. and D.G. carried out bioinformatics analyses; M.J. overproduced and purified all recombinant proteins, and conducted acyl transfer assays and LC–MS analyses of intact proteins; P.P. synthesized the trans-β-chloroacrylamide-containing pantetheine analog, and P.P. and M.J. carried out the crosslinking experiments; S.K. and J.R.L. conducted the NMR and CD experiments, and analyzed the data; A.S.B. and J.E.M. synthesized the diazirine reagent used in the footprinting experiments; L.M. and N.J.O. carried out the footprinting experiments and analyzed the data; M.J., S.K., J.R.L. and G.L.C. wrote the paper, and all authors contributed to revision of the manuscript.

Corresponding authors

Correspondence to Matthew Jenner or Gregory L Challis.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Tables 1–3, Supplementary Figures 1–24 (PDF 4891 kb)

Life Sciences Reporting Summary (PDF 129 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Jenner, M., Kosol, S., Griffiths, D. et al. Mechanism of intersubunit ketosynthase–dehydratase interaction in polyketide synthases. Nat Chem Biol 14, 270–275 (2018). https://doi.org/10.1038/nchembio.2549

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nchembio.2549

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing