Molecular impact of covalent modifications on nonribosomal peptide synthetase carrier protein communication

Andrew C Goodrich,David J Meyers,Dominique P Frueh

doi:10.1074/jbc.m116.766220

Andrew C Goodrich, David J Meyers + Show 1 more

Open Access

https://doi.org/10.1074/jbc.m116.766220

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Abstract

Nonribosomal peptide synthesis involves the interplay between covalent protein modifications, conformational fluctuations, catalysis, and transient protein-protein interactions. Delineating the mechanisms involved in orchestrating these various processes will deepen our understanding of domain-domain communication in nonribosomal peptide synthetases (NRPSs) and lay the groundwork for the rational reengineering of NRPSs by swapping domains handling different substrates to generate novel natural products. Although many structural and biochemical studies of NRPSs exist, few studies have focused on the energetics and dynamics governing the interactions in these systems. Here, we present detailed binding studies of an adenylation domain and its partner carrier protein in apo-, holo-, and substrate-loaded forms. Results from fluorescence anisotropy, isothermal titration calorimetry, and NMR titrations indicated that covalent modifications to a carrier protein modulate domain communication, suggesting that chemical modifications to carrier proteins during NRPS synthesis may impart directionality to sequential NRPS domain interactions. Comparison of the structure and dynamics of an apo-aryl carrier protein with those of its modified forms revealed structural fluctuations induced by post-translational modifications and mediated by modulations of protein dynamics. The results provide a comprehensive molecular description of a carrier protein throughout its life cycle and demonstrate how a network of dynamic residues can propagate the molecular impact of chemical modifications throughout a protein and influence its affinity toward partner domains.

Highlights

Nonribosomal peptide synthesis involves the interplay between covalent protein modifications, conformational fluctuations, catalysis, and transient protein-protein interactions
The results provide a comprehensive molecular description of a carrier protein throughout its life cycle and demonstrate how a network of dynamic residues can propagate the molecular impact of chemical modifications throughout a protein and influence its affinity toward partner domains
We found that chemical modifications to carrier proteins (CPs) can modulate the affinity and thermodynamics of the interactions

Summary

Introduction

Nonribosomal peptide synthesis involves the interplay between covalent protein modifications, conformational fluctuations, catalysis, and transient protein-protein interactions. We present detailed binding studies of an adenylation domain and its partner carrier protein in apo-, holo-, and substrate-loaded forms. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. NRPSs employ a modular, multidomain architecture and a series of enzymatic reactions to covalently tether simple substrates to so-called carrier proteins (CPs) and assemble them into natural products via peptide bonds [3, 4]. A condensation or cyclization domain catalyzes peptide bond formation between the substrate of an upstream CP and that of a downstream CP. Peptide bond formation restores the upstream carrier protein to its holo-form, whereas the downstream CP harbors an elongated intermediate. A thioesterase domain typically releases the product from the terminal carrier protein, either by hydrolysis or macrocyclization

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