Abstract
Nonribosomal peptide synthetases (NRPSs) employ multiple domains separated by linker regions to incorporate substrates into natural products. During synthesis, substrates are covalently tethered to carrier proteins that translocate between catalytic partner domains. The molecular parameters that govern translocation and associated linker remodeling remain unknown. Here, we used NMR to characterize the structure, dynamics, and invisible states of a peptidyl carrier protein flanked by its linkers. We showed that the N-terminal linker stabilizes and interacts with the protein core while modulating dynamics at specific sites involved in post-translational modifications and/or domain interactions. The results detail the molecular communication between peptidyl carrier proteins and their linkers and could guide efforts in engineering NRPSs to obtain new pharmaceuticals.