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Structural basis for intermodular communication in assembly-line polyketide biosynthesis

Nature Chemical Biology (2024)Cite this article

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Abstract

Assembly-line polyketide synthases (PKSs) are modular multi-enzyme systems with considerable potential for genetic reprogramming. Understanding how they selectively transport biosynthetic intermediates along a defined sequence of active sites could be harnessed to rationally alter PKS product structures. To investigate functional interactions between PKS catalytic and substrate acyl carrier protein (ACP) domains, we employed a bifunctional reagent to crosslink transient domain–domain interfaces of a prototypical assembly line, the 6-deoxyerythronolide B synthase, and resolved their structures by single-particle cryogenic electron microscopy (cryo-EM). Together with statistical per-particle image analysis of cryo-EM data, we uncovered interactions between ketosynthase (KS) and ACP domains that discriminate between intra-modular and inter-modular communication while reinforcing the relevance of conformational asymmetry during the catalytic cycle. Our findings provide a foundation for the structure-based design of hybrid PKSs comprising biosynthetic modules from different naturally occurring assembly lines.

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Fig. 1: Crosslinking a PKS module with DBA.
Fig. 2: DBA crosslinking a KS–AT core with its upstream and downstream ACP partners.
Fig. 3: Cryo-EM structure of an intermodular docking interaction during polyketide translocation.
Fig. 4: Structural analysis of crosslinked DEBS M3TE in complex with Fab 1B2 (CL-M3TE-1B2).
Fig. 5: Measuring the ACP occupancy of individual particles by statistical cryo-EM image analysis.

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Data availability

All atomic coordinates and cryo-EM maps have been deposited in the Protein Data Bank (PDB) under accession codes 8TJN, 8TJO, 8TPW, 8TPX, 8TJP and 8TKO and in the Electron Microscopy Data Bank under accession codes EMD-41305, EMD-41306, EMD-41495, EMD-41496, EMD-41307 and EMD-41355 (also declared in the authors’ Reporting Summary). Coordinates for model building were obtained from the PDB via accession codes 7M7F, 2JU2 and 6C9U. All materials used in this study that are not commercially available will be made available by the authors upon reasonable request. Source data are provided with this paper.

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Acknowledgements

We would like to thank W. Chiu (Stanford University) for helpful discussions during the preparation of this manuscript. This study was funded by National Institutes of Health grant R35GM141799 (C.K.); National Institutes of Health grant F32GM136039 (D.P.C.); National Science Foundation Graduate Research Fellowship grant DGE-1656518 (A.M.S); and National Institutes of Health grant R01GM150905 (M.C.). Cryo-EM was performed at the Stanford-SLAC Cryo-EM Center, which is supported by the National Institutes of Health Common Fund Transformative High-Resolution Cryo-Electron Microscopy program (U24GM129541) and the Chan Zuckerberg Initiative (2021-234593).

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Author notes

  1. These authors contributed equally: Alexander M. Soohoo, Muyuan Chen.

Authors and Affiliations

  1. Department of Chemistry, Stanford University, Stanford, CA, USA

    Dillon P. Cogan, Krystal L. Brodsky & Chaitan Khosla

  2. Department of Pharmacology and Pharmaceutical Sciences, University of Southern California, Los Angeles, CA, USA

    Dillon P. Cogan

  3. Department of Chemical Engineering, Stanford University, Stanford, CA, USA

    Alexander M. Soohoo & Chaitan Khosla

  4. Division of CryoEM and Bioimaging, Stanford Synchrotron Radiation Lightsource, SLAC National Accelerator Laboratory, Stanford University, Menlo Park, CA, USA

    Muyuan Chen & Yan Liu

  5. Stanford ChEM-H, Stanford, CA, USA

    Chaitan Khosla

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Contributions

D.P.C., A.M.S., M.C. and C.K. conceived of the project aims. D.P.C. and A.M.S. collected non-cryo-EM experimental data. D.P.C. and Y.L. performed single-particle cryo-EM analysis. D.P.C. and K.L.B. refined the atomic models. M.C. performed the statistical per-particle image analysis. C.K. supervised all experiments. D.P.C. and C.K. wrote the initial manuscript, which was revised and edited by all authors.

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Correspondence to Dillon P. Cogan or Chaitan Khosla.

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Protein sequences, Supplementary Methods, Supplementary Figs. 1–39, source data for supplementary figures, Supplementary Tables 1–11 and Supplementary References.

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Cogan, D.P., Soohoo, A.M., Chen, M. et al. Structural basis for intermodular communication in assembly-line polyketide biosynthesis. Nat Chem Biol (2024). https://doi.org/10.1038/s41589-024-01709-y

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