Structural basis for dimerization, catalytic regulation, and substrate selectivity in S9D proteases

S9 proteases are widely distributed across the tree-of-life and play essential roles in protein processing. However, the structural and mechanistic basis for protease activity in the S9D subfamily has remained unknown. Here, we report the first high-resolution cryo-EM structures of an S9D protease, chloroplast glutamyl endopeptidase (CGEP) from <i>Arabidopsis thaliana</i>, expressed in plants and bacteria. CGEP adopts a dimeric architecture stabilized by two distinct interfaces: hydrophobic interactions between catalytic domains and an interdomain β-sheet linking the cap and catalytic domains. These interactions create a rigid scaffold that supports a hinge loop, which acts as a steric gate to restrict substrate access and confine catalytic activity to the closed conformation. Unlike S9A-B-C proteases, CGEP maintains an intact catalytic triad in both open and closed states, relying on hinge-loop gating rather than catalytic disruption for regulation. Structural analysis and mutagenesis reveal that the hinge loop forms a conserved pocket favoring glutamate side chains, explaining CGEP's strong glutamate preference at cleavage sites. Together, these findings uncover a unique regulatory paradigm for S9D proteases and provide a structural framework for understanding substrate selectivity and dimerization.