Essential Role of Loop Dynamics in Type II NRPS Biomolecular Recognition

• Published in: ACS chemical biology 2022-10, Vol.17 (10), p.2890-2898
• Main Authors: Corpuz, Joshua C., Patel, Ashay, Davis, Tony D., Podust, Larissa M., McCammon, J. Andrew, Burkart, Michael D.
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Carrier Proteins - metabolism; Catalytic Domain; Peptide Synthases - metabolism; Peptides - chemistry
• ISSN: 1554-8929; 1554-8937
• DOI: 10.1021/acschembio.2c00523

Non-ribosomal peptides play a critical role in the clinic as therapeutic agents. To access more chemically diverse therapeutics, non-ribosomal peptide synthetases (NRPSs) have been targeted for engineering through combinatorial biosynthesis; however, this has been met with limited success in part due to the lack of proper protein–protein interactions between non-cognate proteins. Herein, we report our use of chemical biology to enable X-ray crystallography, molecular dynamics (MD) simulations, and biochemical studies to elucidate binding specificities between peptidyl carrier proteins (PCPs) and adenylation (A) domains. Specifically, we determined X-ray crystal structures of a type II PCP crosslinked to its cognate A domain, PigG and PigI, and of PigG crosslinked to a non-cognate PigI homologue, PltF. The crosslinked PCP-A domain structures possess large protein–protein interfaces that predominantly feature hydrophobic interactions, with specific electrostatic interactions that orient the substrate for active site delivery. MD simulations of the PCP-A domain complexes and unbound PCP structures provide a dynamical evaluation of the transient interactions formed at PCP-A domain interfaces, which confirm the previously hypothesized role of a PCP loop as a crucial recognition element. Finally, we demonstrate that the interfacial interactions at the PCP loop 1 region can be modified to control PCP binding specificity through gain-of-function mutations. This work suggests that loop conformational preferences and dynamism account for improved shape complementary in the PCP-A domain interactions. Ultimately, these studies show how crystallographic, biochemical, and computational methods can be used to rationally re-engineer NRPSs for non-cognate interactions.