Carrier Protein Interaction with Competing Adenylation and Epimerization Domains in a Nonribosomal Peptide Synthetase Analyzed by FRET.

In multi-domain nonribosomal peptide synthetases (NRPSs) the order of domains and their catalytic specificities dictate the structure of the peptide product. Peptidyl-carrier proteins (PCPs) bind activated amino acids and channel elongating peptidyl intermediates along the protein template. To this end, fine-tuned interactions with the catalytic domains and large-scale PCP translocations are necessary. Despite crystal structure snapshots of several PCP-domain interactions, the conformational dynamics under catalytic conditions in solution remain poorly understood. We report a FRET reporter of gramicidin S synthetase 1 (GrsA; with A-PCP-E domains) to study for the first time the interaction between PCP and adenylation (A) domain in the presence of an epimerization (E) domain, a competing downstream partner for the PCP. Bulk FRET measurements showed that upon PCP aminoacylation a conformational shift towards PCP binding to the A domain occurs, indicating the E domain acts on its PCP substrate out of a disfavored conformational equilibrium. Furthermore, the A domain was found to preferably bind the D-Phe-S-Ppant-PCP stereoisomer, suggesting it helps in establishing the stereoisomeric mixture in favor of the D-aminoacyl moiety. These observations surprisingly show that the conformational logic can deviate from the order of domains and thus reveal new principles in the multi-domain interplay of NRPSs.

Subdomain dynamics enable chemical chain reactions in non-ribosomal peptide synthetases.

Many peptide-derived natural products are produced by non-ribosomal peptide synthetases (NRPSs) in an assembly-line fashion. Each amino acid is coupled to a designated peptidyl carrier protein (PCP) through two distinct reactions catalysed sequentially by the single active site of the adenylation domain (A-domain). Accumulating evidence suggests that large-amplitude structural changes occur in different NRPS states; yet how these molecular machines orchestrate such biochemical sequences has remained elusive. Here, using single-molecule Förster resonance energy transfer, we show that the A-domain of gramicidin S synthetase I adopts structurally extended and functionally obligatory conformations for alternating between adenylation and thioester-formation structures during enzymatic cycles. Complementary biochemical, computational and small-angle X-ray scattering studies reveal interconversion among these three conformations as intrinsic and hierarchical where intra-A-domain organizations propagate to remodel inter-A-PCP didomain configurations during catalysis. The tight kinetic coupling between structural transitions and enzymatic transformations is quantified, and how the gramicidin S synthetase I A-domain utilizes its inherent conformational dynamics to drive directional biosynthesis with a flexibly linked PCP domain is revealed.

FRET Monitoring of a Nonribosomal Peptide Synthetase Elongation Module Reveals Carrier Protein Shuttling between Catalytic Domains.

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.

Intermediary conformations linked to the directionality of the aminoacylation pathway of nonribosomal peptide synthetases.

Nonribosomal peptide synthetases (NRPSs) are multifunctional megaenzymes that govern the stepwise biosynthesis of pharmaceutically important peptides. In an ATP-dependent assembly-line mechanism dedicated domains are responsible for each catalytic step. Crystal structures have provided insight into several conformations of interacting domains. However, the complete picture in solution of how domain dynamics and the timing of conformational changes effect a directional biosynthesis remains only poorly understood and will be important for the efficient reprogramming of NRPSs. Here we dissect the multiple conformational changes associated with the adenylation and thiolation reactions of the aminoacylation pathway under catalytic conditions. We used pyrophosphate (PP i ) to biochemically drive the conformational changes backward and forward while performing an online monitoring with a Förster resonance energy transfer (FRET) didomain sensor, consisting of adenylation (A) and peptidyl-carrier protein (PCP) domains. Notably, we found aminoacyl thioester formation to efficiently occur in the presence of PP i even at millimolar concentrations, despite the chemically and conformationally reversing effect of this metabolite and by-product. This finding settles conflicting reports and explains why intracellular PP i concentrations do not impair NRP biosynthesis. A conserved amino acid was identified to be important for the mechanism under these conditions. FRET time-course analyses revealed that the directionality of the aminoacylation catalysis is correlated with conformational kinetics. Complemented by equilibrium hydrogen-deuterium exchange (HDX) analyses, our data uncovered the existence of at least one new intermediary conformation that is associated with the rate-determining step. We propose an expanded model of conformational changes in the NRPS aminoacylation pathway.

Context-dependent activity of A domains in the tyrocidine synthetase.

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.

FRET monitoring of a nonribosomal peptide synthetase.

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.