Nonribosomal peptide synthetases (NRPSs) are multidomain enzyme templates for the synthesis of bioactive peptides. Large-scale conformational changes during peptide assembly are obvious from crystal structures, yet their dynamics and coupling to catalysis are poorly understood. We have designed an NRPS FRET sensor to monitor, in solution and in real time, the adoption of the productive transfer conformation between phenylalanine-binding adenylation (A) and peptidyl-carrier-protein domains of gramicidin synthetase I from Aneurinibacillus migulanus. The presence of ligands, substrates or intermediates induced a distinct fluorescence resonance energy transfer (FRET) readout, which was pinpointed to the population of specific conformations or, in two cases, mixtures of conformations. A pyrophosphate switch and lysine charge sensors control the domain alternation of the A domain. The phenylalanine-thioester and phenylalanine-AMP products constitute a mechanism of product inhibition and release that is involved in ordered assembly-line peptide biosynthesis. Our results represent insights from solution measurements into the conformational dynamics of the catalytic cycle of NRPSs.

In-solution analysis of conformational changes of NRPS adenylation and peptidyl-carrier protein domains under catalytic conditions reveals a new intermediary conformation.

Non-ribosomal peptide synthetases (NRPSs) are large, modular enzymes that produce bioactive peptides of tremendous structural and chemical diversity, due to the incorporation, alongside the canonical 20 amino acids, of non-proteinogenic amino acids, fatty acids, sugars and heterocyclic rings. For linear NRPSs, the size and composition of the peptide product is dictated by the number, order and specificity of the individual modules, each made of several domains. Given the size and complexity of NRPSs, most in vitro studies have focused on individual domains, di-domains or single modules extracted from the full-length proteins. However, intermodular interactions could play a critical role and regulate the activity of the domains and modules in unpredictable ways. Here we investigate in vitro substrate activation by three A domains of the tyrocidine synthetase TycC enzyme, systematically comparing their activity when alone (with the respective PCP domain), in pairs (di-modular constructs) or all together (tri-modular construct). Furthermore, we study the impact of mutations in the A or PCP domains in these various constructs. Our results suggest that substrate adenylation and effects of mutations largely depend on the context in which the domains/modules are. Therefore, generalizing properties observed for domains or modules in isolation should be done with caution.

Nonribosomal peptide synthetases (NRPSs) employ multiple domains, specifically arranged in modules, for the assembly-line biosynthesis of a plethora of bioactive peptides. It is poorly understood how catalysis is correlated with the domain interplay and associated conformational changes. We developed FRET sensors of an elongation module to study in solution the intramodular interactions of the peptidyl carrier protein (PCP) with adenylation (A) and condensation (C) domains. Backed by HDX-MS analysis, we discovered dynamic mixtures of conformations that undergo distinct population changes in favor of the PCP-A and PCP-C interactions upon completion of the adenylation and thiolation reactions, respectively. To probe this model we blocked PCP binding to the C domain by photocaging and triggered peptide bond formation with light. Changing intramodular domain affinities of the PCP appear to result in conformational shifts according to the logic of the templated assembly process.

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