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 Arabidopsis thaliana, expressed in plants and bacteria. CGEP adopts a dimeric architecture stabilized by two distinct interfaces: hydrophobic interactions between catalytic domains and an interdomain {beta}-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 CGEPs 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. Significance statementThis study provides the first structural and mechanistic insights into plant S9D proteases, revealing a unique regulatory paradigm that combines hinge-loop gating with substrate-specific recognition. By uncovering how CGEP maintains catalytic integrity while restricting activity to the closed conformation and explaining its strong glutamate preference, these findings advance our understanding of protease diversity and open new avenues for engineering proteases with tailored specificity for agricultural and biotechnological applications.