1 research outputs found
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<sub>BS</sub>) 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<sub>BS</sub> self-processes its own C-termini. We have determined the crystal
structure of a proteolytically stable fragment of SppA<sub>BS</sub>K199A 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<sub>BS</sub> competes with a fluorometric peptide
substrate for the SppA<sub>BS</sub> 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