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.
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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.
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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.