S27: Using small molecule probes to study the functional and structural interaction of adenylation and carrier protein domains in nonribosomal peptide synthetases

Nonribosomal peptide synthetases (NRPSs) are multidomain proteins that catalyze the synthesis of a wide array of fascinating complex peptide natural products including the antibiotics vancomycin and daptomycin as well as the bacterial siderophores such as enterobactin and mycobactin.  NRPSs contain three core domains: 1) an adenylation domain that catalyzes a two-step adenylation–acylation of an amino acid; 2) a thiolation or carrier protein domain onto which the amino acid is covalently tethered as a thioester, and 3) a condensation domain that catalyzes peptide bond formation between adjacent aminothioacyl loaded carrier protein domains.  The conformational flexibility of these large multidomain proteins has hindered structural efforts to characterize catalytically relevant interdomain interactions of these modular enzymes. We will describe the design, synthesis, and application of small molecule mechanism-based inhibitors to foster crystallization of several representative multidomain NRPS that has provided detailed structural insight into adenylation-carrier protein domain interactions.