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

DOI: 10.1021/acscatal.1c02113 DOI Help

Authors: Christopher D. Fage (Philipps-Universität Marburg; University of Warwick) , Simone Kosol (University of Warwick) , Matthew Jenner (University of Warwick) , Carl Öster (University of Warwick) , Angelo Gallo (University of Warwick) , Milda Kaniusaite (Monash University) , Roman Steinbach (Philipps-Universität Marburg) , Michael Staniforth (University of Warwick) , Vasilios G. Stavros (University of Warwick) , Mohamed A. Marahiel (Philipps-Universität Marburg) , Max J. Cryle (Monash University) , Józef R. Lewandowski (University of Warwick)
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Acs Catalysis

State: Published (Approved)
Published: August 2021
Diamond Proposal Number(s): 16655

Open Access Open Access

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.

Journal Keywords: biosynthesis; docking domain; intrinsically disordered protein; natural product; nonribosomal peptide synthetase

Diamond Keywords: Enzymes

Subject Areas: Biology and Bio-materials, Chemistry

Instruments: B23-Circular Dichroism

Other Facilities: ID23-1 at ESRF

Added On: 18/08/2021 09:10


Discipline Tags:

Biochemistry Catalysis Chemistry Biophysics Life Sciences & Biotech

Technical Tags:

Spectroscopy Circular Dichroism (CD)