What role does intra-domain protein dynamics play in multi-domain protein communication? We answer this from investigations of allosteric communication in a peptide-bond catalyzing domain of a nonribosomal peptide synthetase (NRPS). NRPSs are modular, multidomain enzymatic systems that synthesize complex natural products from simple substrates, many with invaluable pharmacological applications (antibiotics like bacitracin, immunosuppressants like rapamycin, etc.). NRPSs operate in a remarkably conserved assembly-line like manner, with each NRPS module elongating the product by one substrate through transient sequential interactions between peptide-bond forming condensation (C) domains and substrate-presenting carrier protein (CP) domains. During synthesis, the substrates and growing products are tethered to CP prosthetic arms. Whether and how these sequential interactions are orchestrated during synthesis remains unknown. Our investigations reveal that intra-domain dynamics drives inter-domain interactions. Using solution NMR, we discovered that structural dynamics within a C-like domain connect remote binding sites for upstream and downstream CPs. This dynamic pathway senses substrate on the upstream CP, promotes binding between the C and CP domains, and induces an allosteric response to the remote binding site of the downstream CP. We demonstrate that a single mutation that impedes intra-domain dynamics also severes the allosteric response and recognition of substrates on the partner CP, thus unequivocally linking intra-domain dynamic allostery to inter-domain communication. This mutation in turn uncovered a conserved feature maintained in C-domains and related C-like domains. Our results demonstrate that conserved dynamics within members of the C-domain family choreograph inter-domain NRPS dynamics during synthesis, a feature that should be maintained when re-engineering NRPSs to make new products.

Dynamic allostery in condensation-like domains drives communication in nonribosomal peptide synthetases

ACS Spring 2022

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