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Communication breakdown : dissecting the COM interfaces between the subunits of nonribosomal peptide synthetases

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Fage, Christopher, Kosol, Simone, Jenner, Matthew, Öster, Carl, Gallo, Angelo, Kaniusaite, Milda, Steinbach, Roman, Staniforth, Michael, Stavros, Vasilios G., Marahiel, Mohamed A., Cryle, Max J. and Lewandowski, Józef R. (2021) Communication breakdown : dissecting the COM interfaces between the subunits of nonribosomal peptide synthetases. ACS Catalysis, 11 . pp. 10802-10813. doi:10.1021/acscatal.1c02113

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Official URL: http://dx.doi.org/10.1021/acscatal.1c02113

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Abstract

Nonribosomal peptides are a structurally diverse and bioactive class of natural products constructed by multidomain enzymatic assembly lines known as nonribosomal peptide synthetases (NRPSs). While the core catalytic domains and even entire protein subunits of NRPSs have been structurally elucidated, little biophysical work has been reported on the docking domains that promote interactions—and thus transfer of biosynthetic intermediates—between subunits. In the present study, we closely examine the COM domains that mediate COMmunication between donor epimerization (E) and acceptor condensation (C) domains found at the termini of NRPS subunits. Through a combination of X-ray crystallography, circular dichroism spectroscopy, solution- and solid-state NMR spectroscopy, and molecular dynamics (MD) simulations, we provide direct evidence for an intrinsically disordered donor COM region that folds into a dynamic helical motif upon binding to a suitable acceptor. Furthermore, our NMR titration and carbene footprinting experiments illuminate the residues involved at the COM interaction interface, and our MD simulations demonstrate folding consistent with experimental data. Although our results lend credence to the previously proposed helix-hand mode of interaction, they also underscore the importance of viewing COM interfaces as dynamic ensembles rather than single rigid structures and suggest that engineering experiments should account for the interactions which transiently guide folding in addition to those which stabilize the final complex. Through activity assays and affinity measurements, we further substantiate the role of the donor COM region in binding the acceptor C domain and implicate this short motif as readily transposable for noncognate domain crosstalk. Finally, our bioinformatics analyses show that COM domains are widespread in natural product pathways and function at interfaces beyond the canonical type described above, setting a high priority for thorough characterization of these docking domains. Our findings lay the groundwork for future attempts to rationally engineer NRPS domain–domain interactions with the ultimate goal of generating bioactive molecules.

Item Type: Journal Article
Subjects: Q Science > QH Natural history
Q Science > QP Physiology
Divisions: Faculty of Science > Chemistry
Library of Congress Subject Headings (LCSH): Biosynthesis , Protein-protein interactions, Proteins -- Structure, Proteins -- Conformation, Natural products , Natural products -- Biotechnology, Peptides -- Synthesis
Journal or Publication Title: ACS Catalysis
Publisher: American Chemical Society
ISSN: 2155-5435
Official Date: 16 August 2021
Dates:
DateEvent
16 August 2021Published
16 August 2021Accepted
Volume: 11
Page Range: pp. 10802-10813
DOI: 10.1021/acscatal.1c02113
Status: Peer Reviewed
Publication Status: Published
Access rights to Published version: Open Access
Copyright Holders: © 2021 The Authors. Published by American Chemical Society
RIOXX Funder/Project Grant:
Project/Grant IDRIOXX Funder NameFunder ID
UNSPECIFIED[DFG] Deutsche Forschungsgemeinschafthttp://dx.doi.org/10.13039/501100001659
EP/M027503/1[EPSRC] Engineering and Physical Sciences Research Councilhttp://dx.doi.org/10.13039/501100000266
FP/2007- 2013; 639907Seventh Framework Programmehttp://dx.doi.org/10.13039/100011102
BB/L022761/1[BBSRC] Biotechnology and Biological Sciences Research Councilhttp://dx.doi.org/10.13039/501100000268
BB/R010218/1[BBSRC] Biotechnology and Biological Sciences Research Councilhttp://dx.doi.org/10.13039/501100000268
BB/R012121/1[BBSRC] Biotechnology and Biological Sciences Research Councilhttp://dx.doi.org/10.13039/501100000268
BB/M017982/1[BBSRC] Biotechnology and Biological Sciences Research Councilhttp://dx.doi.org/10.13039/501100000268
316630Seventh Framework Programmehttp://dx.doi.org/10.13039/100011102
EP/N010825/1[EPSRC] Engineering and Physical Sciences Research Councilhttp://dx.doi.org/10.13039/501100000266
EP/N010825/1[EPSRC] Engineering and Physical Sciences Research Councilhttp://dx.doi.org/10.13039/501100000266
UNSPECIFIEDRoyal Societyhttp://dx.doi.org/10.13039/501100000288
UNSPECIFIEDLeverhulme Trusthttp://dx.doi.org/10.13039/501100000275
APP1140619 National Health and Medical Research Councilhttp://dx.doi.org/10.13039/501100000925
DP190101272 Australian Research Councilhttp://dx.doi.org/10.13039/501100000923
CE200100012Australian GovernmentUNSPECIFIED
SM16655Diamond Light Sourcehttp://dx.doi.org/10.13039/100011889

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