Conformational remodeling enhances activity of lanthipeptide zinc-metallopeptidases

Abstract

Lanthipeptides are an important group of natural products with diverse biological functions, and their biosynthesis requires the removal of N-terminal leader peptides (LPs) by designated proteases. LanP_M1 enzymes, a subgroup of M1 zinc-metallopeptidases, have been recently identified as bifunctional proteases with both endo- and aminopeptidase activities to remove LPs of class III and class IV lanthipeptides. Herein, we report the biochemical and structural characterization of EryP as the LanP_M1 enzyme from the biosynthesis of class III lanthipeptide erythreapeptin. We determined X-ray crystal structures of EryP in three conformational states, the open, intermediate and closed states, and identified a unique interdomain Ca²⁺ binding site as a regulatory element that modulates its domain dynamics and proteolytic activity. Inspired by this regulatory Ca²⁺ binding, we developed a strategy to engineer LanP_M1 enzymes for enhanced catalytic activities by strengthening interdomain associations and driving the conformational equilibrium toward their closed forms.