Analysis of the linker region joining the adenylation and carrier protein domains of modular non-ribosomal peptide synthetases

Combinatorial biosynthesis of Non-Ribosomal Peptide Synthetases (NRPSs) is a unique approach for the development of novel peptide natural products that may have therapeutic benefits. The replacement of domains within a NRPS module to change the overall natural product is met with difficulties due to incompatibilities between interacting domains. Recent crystal structures of multi-domain NRPSs give insight into how these large enzymes interact and the conformational changes necessary to carry out natural product biosynthesis. Specifically we now know how the various domains interact and are positioned to examine the role of the peptide linkers that connect them. Using a bioinformatic, biochemical, and structural approach, we demonstrate that the adenylation-peptidyl carrier protein linker of multi-domain NRPSs consists of several proline residues not found in standalone adenylation domains. While some of these prolines may impart rigidity to the coiled linker, prolines located at the beginning of the linker form a conserved LPxP motif not seen in standalone adenylation domains. This motif, which replaces an alpha helix seen in standalone adenylation domains, anchors the linker to the C-terminal sub-domain of the adenylation domain through interactions with a conserved tyrosine. This interaction subsequently stabilizes the A10 motif for acyl-adenylate formation and provides a potential link between a large conformational change within the adenylation domain and the proper delivery of the peptidyl carrier protein.