The role of constitutively inactive isoforms of GrsA in regulating the initiation cascade of gramicidin synthesis

Abstract

Abstract Non-ribosomal Peptide Synthetases (NRPSs) provide exciting targets for templated drug-design because of their assembly-line like mechanism of action. The non-catalytic conformations of the NRPS, GrsA, are proposed to regulate the activation cascade during the synthesis of the antibiotic peptide, Gramicidin. Previous studies hinted at the possibilities of additional, uncharacterized intermediates from the initiation cascade. Here, we redefined the mechanism of catalytic cycle and identified two new structural intermediates showing ‘near-thiolation’ events. The first structural intermediate formed without the need for dissociation of PPi. HDX-MS also revealed a ‘product-release’ intermediate attained solely from the intramolecular allosteric effects of substrate binding at the Adenylation domain. SAXS allowed us to determine the full-length envelope of GrsA for the first time and showed that its resting state is comprised of two inactive conformations. Calorimetry and molecular dynamics simulations provided further evidence supporting a constitutively inactive regulatory framework opening a new paradigm of NRPS research.