Conformational remodeling enhances activity of lanthipeptide zinc-metallopeptidases.

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 Ca 2+ binding site as a regulatory element that modulates its domain dynamics and proteolytic activity. Inspired by this regulatory Ca 2+ 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.