Structure of Signal Peptide Peptidase A with C‑Termini Bound in the Active Sites: Insights into Specificity, Self-Processing, and Regulation

Bacterial signal peptide peptidase A (SppA) is a membrane-bound enzyme that utilizes a serine/lysine catalytic dyad mechanism to cleave remnant signal peptides within the cellular membrane. <i>Bacillus subtilis</i> SppA (SppA_BS) oligomerizes into a homo-octameric dome-shaped complex with eight active sites, located at the interface between each protomer. In this study, we show that SppA_BS self-processes its own C-termini. We have determined the crystal structure of a proteolytically stable fragment of SppA_BSK199A that has its C-terminal peptide bound in each of the eight active sites, creating a perfect circle of peptides. Substrate specificity pockets S1, S3, and S2′ are identified and accommodate C-terminal residues Tyr331, Met329, and Tyr333, respectively. Tyr331 at the P1 position is conserved among most <i>Bacillus</i> species. The structure reveals that the C-terminus binds within the substrate-binding grooves in an antiparallel β-sheet fashion. We show, by C-terminal truncations, that the C-terminus is not essential for oligomeric assembly. Kinetic analysis shows that a synthetic peptide corresponding to the C-terminus of SppA_BS competes with a fluorometric peptide substrate for the SppA_BS active site. A model is proposed for how the C-termini of SppA may function in the regulation of this membrane-bound self-compartmentalized protease