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

Abstract

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.

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

“…Obviously, the effective intramolecular affinity of the A domain towards the aminoacyl‐PCP is high enough to win the competition with the E domain at least to a significant proportion. Interestingly, this finding is in stark contrast to a previously reported domain interplay in the TycB1 elongation module (with C‐A‐PCP domains ) in which the acceptor site of the C domain represents the second competing downstream binding partner of the PCP, next to the A domain [13c] . For this module, upon aminoacylation, the PCP was found to preferably bind to the C domain, as detected by a depopulation of the transfer conformation and a conformational shift towards the C conformation [13c] .…”

“…In contrast, the structural behavior of NRPSs in solution is still underexplored but crucial to understand relevant conformations and the temporal and spatial dynamics associated with their interconversions during catalysis. To address these issues, we have previously studied NRPS conformations in solution [12] as well as under catalytic conditions using Förster resonance energy transfer (FRET) [13] and Hydrogen‐Deuterium exchange mass spectrometry (HDX‐MS) [13b,c] analyses. A FRET sensor based on a truncated A‐PCP didomain construct of the GrsA (Figure 1A) served to study the A↔PCP interaction [13a,b] .…”

“…Rather, the aminoacylated PCP is required to interact with the downstream catalytic domains. It thus remains unclear whether the observed back‐binding to the A domain [13a,b] indeed represents the conformational behavior of the aminoacylated protein or if it was an artificial result due to the missing downstream domain in the truncated A‐PCP sensor construct [13c] …”

Structural advances toward understanding the catalytic activity and conformational dynamics of modular nonribosomal peptide synthetases

“…This feature of the NRPS module is further supported by a series of elegant studies that have used biophysical approaches to explore the dynamics and equilibrium of the core domains. In these studies, [185][186][187] small molecule uorescent probes and uorescent protein domains were employed to monitor via uorescence energy transfer experiments the relative orientations of NRPS domains in solution. Studies with a adenylation PCP didomain support the formation of the two catalytic conformations supporting the domain alternation hypothesis, 38 noting that addition of excess PPi drives the A sub towards the adenylate-forming conformation.…”

“…Natural Product Reports peptide. 186 Inclusion of a downstream condensation domain in a C-A-PCP-C, or indeed the inclusion of a full downstream module, while technically challenging, will undoubtedly offer more insight into the equilibrium of the loaded PCP between three potential catalytic domains.…”