Structural Basis for a Scaffolding Role of the COM Domain in Nonribosomal Peptide Synthetases

Nonribosomal peptide synthetases (NRPSs) are multi-domain enzymes that catalyze the biosynthesis of therapeutically relevant natural products. Efficient peptide synthesis relies on intricate domain interactions, whose underlying principles remain poorly understood. The communication-mediating (COM) domains facilitate interactions between separate NRPS subunits. For unknown reasons, COM domains co-occur with epimerization (E) domains, are partially embedded within the adjacent condensation (C) domains and can also be found as internal cis-COM domains. These features set COM domains apart from other docking domains. We present the first crystal structure of a cis-COM domain within an E-COM-C domain arrangement from modules 4 and 5 of bacitracin synthetase 3 (BacC). The structure reveals a compactly folded COM domain sandwiched between E and C domains, suggesting a role of the COM domain in orienting these domains for efficient peptidyl carrier protein (PCP) shuttling. Through mutational analyses, dipeptide formation assays, and proximity-dependent photo-crosslinking experiments, we investigated both cis- and trans-COM domains and provide evidence supporting a principal role of COM domains as scaffolds of NRPS architecture. Their function as docking domains may be a secondary consequence of their division into separate donor and acceptor parts.