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

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Abstract
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.

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