Modular polyketide synthases and non-ribosomal peptide synthetases are molecular assembly lines that consist of several multienzyme subunits that undergo dynamic self-assembly to form a functional megacomplex. N- and C-terminal docking domains are usually responsible for mediating the interactions between subunits. Here we show that communication between two non-ribosomal peptide synthetase subunits responsible for chain release from the enacyloxin polyketide synthase, which assembles an antibiotic with promising activity against Acinetobacter baumannii, is mediated by an intrinsically disordered short linear motif and a β-hairpin docking domain. The structures, interactions and dynamics of these subunits were characterized using several complementary biophysical techniques to provide extensive insights into binding and catalysis. Bioinformatics analyses reveal that short linear motif/β-hairpin docking domain pairs mediate subunit interactions in numerous non-ribosomal peptide and hybrid polyketide–non-ribosomal peptide synthetases, including those responsible for assembling several important drugs. Short linear motifs and β-hairpin docking domains from heterologous systems are shown to interact productively, highlighting the potential of such interfaces as tools for biosynthetic engineering.
Subscribe to Journal
Get full journal access for 1 year
only $14.08 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Rent or Buy article
Get time limited or full article access on ReadCube.
All prices are NET prices.
The structures of the Bamb_5917 PCP domain and Bamb_5915 are available from the PDB (accession IDs 5MTI and 6CGO, respectively). NMR assignments for the apo- and holo-Bamb_5917 PCP domain are available from the BMRB (http://www.bmrb.wisc.edu/; accession IDs 34085 and 27304, respectively). Raw NMR and BLI data can be obtained from http://wrap.warwick.ac.uk/123013/. The remaining data supporting the findings of this study are included in the Supplementary Information or are available from the corresponding authors upon request. All biological materials are available from the authors upon request.
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).
Xu, W., Qiao, K. & Tang, Y. Structural analysis of protein–protein interactions in type I polyketide synthases. Crit. Rev. Biochem. Mol. Biol. 48, 98–122 (2013).
Weissman, K. J. & Müller, R. Protein–protein interactions in multienzyme megasynthetases. Chembiochem 9, 826–848 (2008).
Hacker, C. et al. Structure-based redesign of docking domain interactions modulates the product spectrum of a rhabdopeptide-synthesizing NRPS. Nat. Commun. 9, 4366 (2018).
Dowling, D. P. et al. Structural elements of an NRPS cyclization domain and its intermodule docking domain. Proc. Natl Acad. Sci. USA 113, 12432–12437 (2016).
Masschelein, J. et al. A dual transacylation mechanism for polyketide synthase chain release in enacyloxin antibiotic biosynthesis. Nat. Chem. https://doi.org/10.1038/s41557-019-0309-7 (2019).
Mahenthiralingam, E. et al. Enacyloxins are products of an unusual hybrid modular polyketide synthase encoded by a cryptic Burkholderia ambifaria Genomic Island. Chem. Biol. 18, 665–677 (2011).
Leslie, A. G. W. Refined crystal structure of type III chloramphenicol acetyltransferase at 1.75 Å resolution. J. Mol. Biol. 213, 167–186 (1990).
De Crécy-Lagard, V., Marlière, P. & Saurin, W. Multienzymatic non ribosomal peptide biosynthesis: identification of the functional domains catalysing peptide elongation and epimerisation. C.R. Acad. Sci. III 318, 927–936 (1995).
Samel, S. A., Schoenafinger, G., Knappe, T. A., Marahiel, M. A. & Essen, L.-O. Structural and functional insights into a peptide bond-forming bidomain from a nonribosomal peptide synthetase. Structure 15, 781–792 (2007).
Holm, L. & Rosenström, P. Dali server: conservation mapping in 3D. Nucleic Acids Res. 38, W545–W549 (2010).
Richter, C. D., Nietlispach, D., Broadhurst, R. W. & Weissman, K. J. Multienzyme docking in hybrid megasynthetases. Nat. Chem. Biol. 4, 75–81 (2008).
Koglin, A. et al. Conformational switches modulate protein interactions in peptide antibiotic synthetases. Science 312, 273–276 (2006).
Lohman, J. R. et al. The crystal structure of BlmI as a model for nonribosomal peptide synthetase peptidyl carrier proteins. Proteins Struct. Funct. Bioinf. 82, 1210–1218 (2014).
Garcı́a de la Torre, J., Huertas, M. L. & Carrasco, B. HYDRONMR: prediction of NMR Relaxation of Globular Proteins from Atomic-Level Structures and Hydrodynamic Calculations. J. Magn. Reson. 147, 138–146 (2000).
Williamson, M. P. Using chemical shift perturbation to characterise ligand binding. Prog. Nucl. Magn. Reson. Spectrosc. 73, 1–16 (2013).
Davey, N. E. et al. Attributes of short linear motifs. Mol. BioSyst. 8, 268–281 (2012).
Vallurupalli, P., Bouvignies, G. & Kay, L. E. Studying “invisible” excited protein states in slow exchange with a major state conformation. J. Am. Chem. Soc. 134, 8148–8161 (2012).
Manzi, L. et al. Carbene footprinting accurately maps binding sites in protein–ligand and protein–protein interactions. Nat. Commun. 7, 13288 (2016).
Lamley, J. M. et al. Solid-state NMR of a protein in a precipitated complex with a full-length antibody. J. Am. Chem. Soc. 136, 16800–16806 (2014).
Bertini, I. et al. Solid-state NMR of proteins sedimented by ultracentrifugation. Proc. Natl Acad. Sci. USA 108, 10396–10399 (2011).
Mainz, A., Jehle, S., van Rossum, B. J., Oschkinat, H. & Reif, B. Large protein complexes with extreme rotational correlation times investigated in solution by magic-angle-spinning NMR spectroscopy. J. Am. Chem. Soc. 131, 15968–15969 (2009).
Wishart, D. S. Interpreting protein chemical shift data. Prog. Nucl. Magn. Reson. Spectrosc. 58, 62–87 (2011).
Hamelberg, D., Mongan, J. & McCammon, J. A. Accelerated molecular dynamics: a promising and efficient simulation method for biomolecules. J. Chem. Phys. 120, 11919–11929 (2004).
Bloudoff, K., Rodionov, D. & Schmeing, T. M. Crystal structures of the first condensation domain of CDA synthetase suggest conformational changes during the synthetic cycle of nonribosomal peptide synthetases. J. Mol. Biol. 425, 3137–3150 (2013).
Bloudoff, K. & Schmeing, T. M. Structural and functional aspects of the nonribosomal peptide synthetase condensation domain superfamily: discovery, dissection and diversity. Biochim. Biophys. Acta Proteins Proteomics 1865, 1587–1604 (2017).
Bisht, N. K. et al. Ligand migration and hexacoordination in type 1 non-symbiotic rice hemoglobin. Biochim. Biophys. Acta 1814, 1042–1053 (2011).
Van Zundert, G. C. P. et al. The HADDOCK2.2 web server: user-friendly integrative modeling of biomolecular complexes. J. Mol. Biol. 428, 720–725 (2016).
Wassenaar, T. A. et al. WeNMR: structural biology on the grid. J. Grid Comput. 10, 743–767 (2012).
Zhang, J. et al. Structural basis of nonribosomal peptide macrocyclization in fungi. Nat. Chem. Biol. 12, 1001–1003 (2016).
Chen, W. H., Li, K., Guntaka, N. S. & Bruner, S. D. Interdomain and intermodule organization in epimerization domain containing nonribosomal peptide synthetases. ACS Chem. Biol. 11, 2293–2303 (2016).
Medema, M. H. et al. Minimum information about a biosynthetic gene cluster. Nat. Chem. Biol. 11, 625–631 (2015).
Dosztanyi, 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).
Agarwala, R. et al. Database resources of the National Center for Biotechnology Information. Nucleic Acids Res. 45, D12–D17 (2017).
Inahashi, Y. et al. Watasemycin biosynthesis in Streptomyces venezuelae: thiazoline C-methylation by a type B radical-SAM methylase homologue. Chem. Sci. 8, 2823–2831 (2017).
Jenner, M. et al. Mechanism of intersubunit ketosynthase–dehydratase interaction in polyketide synthases. Nat. Chem. Biol. 14, 270–275 (2018).
Cilia, E., Pancsa, R., Tompa, P., Lenaerts, T. & Vranken, W. F. The DynaMine webserver: predicting protein dynamics from sequence. Nucleic Acids Res. 42, W264–W270 (2014).
The European Research Council under the European Union’s Seventh Framework Programme (FP/2007-2013; ERC Grant Agreement 639907) supported this research. J.R.L. acknowledges funding from the Royal Society (RG130022), the EPSRC (EP/L025906/1), the BBSRC (BB/L022761/1 and BB/R010218/1) and the Gates Foundation (OPP1160394). The European Commission (Marie Sklodowska-Curie Fellowship; contract no. 656067) and the Research Foundation Flanders funded J.M. G.L.C. acknowledges the BBSRC (BB/L021692/1 and BB/K002341/1) and the Royal Society (Wolfson Research Merit Award WM130033) for funding. The University of Warwick funded P.K.S. through an Institute of Advanced Study fellowship. D.G. and S.Z. were supported by the EPSRC through the Centre for Doctoral Training in Molecular Analytical Science (EP/L015307/1) and the Bridging the Gaps—EPS and AMR initiative (EP/M027503/1), respectively. E.L.C.S. is a Research Career Development Fellow in the Warwick Integrative Synthetic Biology Centre supported by the BBSRC and EPSRC (BB/M017982/1). We acknowledge the FP7 WeNMR (261572) and H2020 West-Life (675858) European e-Infrastructure projects for the use of their web portals, which make use of the EGI infrastructure and DIRAC4EGI service with the dedicated support of CESNET-MetaCloud, INFN-PADOVA, NCG-INGRID-PT, RAL-LCG2, TW-NCHC, IFCA-LCG2, SURFsara and NIKHEF, and the additional support of the national GRID Initiatives of Belgium, France, Italy, Germany, the Netherlands, Poland, Portugal, Spain, UK, South Africa, Malaysia, Taiwan and the US Open Science Grid. We thank A. Marsh for providing access to the workstation used for the aMD simulation of Bamb_5915. Molecular graphics were generated using UCSF Chimera and Chimera X, developed by the Resource for Biocomputing, Visualization, and Informatics at the University of California, San Francisco, supported by the NIH (P41-GM103311 and R01-GM129325). We thank G. Bouvignies for assistance with ChemEx.
The authors declare no competing interests.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Details of the materials and methods used, Figs. 1–43 and Tables 1–15.
Raw carbene footprinting data for Bamb_5915 and Bamb_5917, and SLiM–βHD domain pair hits from the GenBank database (database accessed July 2018).
Fly through the solvent channel in the X-ray crystal structure of Bamb_5915.
1 µs accelerated MD simulation of Bamb_5915.
Fly through the solvent channel after 0.528 µs aMD simulations of Bamb_5915.
The first mode from Principal Component Analysis of the 1 µs aMD simulations of Bamb_5915.
The first mode from Principal Component Analysis of the 0.5 µs aMD simulations of the holo-Bamb_5917 PCP domain.
About this article
Cite this article
Kosol, S., Gallo, A., Griffiths, D. et al. Structural basis for chain release from the enacyloxin polyketide synthase. Nat. Chem. 11, 913–923 (2019). https://doi.org/10.1038/s41557-019-0335-5
Angewandte Chemie International Edition (2020)
Phytotherapy Research (2020)
Exploring modular reengineering strategies to redesign the teicoplanin non-ribosomal peptide synthetase
Chemical Science (2020)
Impact of Magnetic Field Strength on Resolution and Sensitivity of Proton Resonances in Biological Solids
The Journal of Physical Chemistry C (2020)
Angewandte Chemie (2020)