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. Bacillus subtilis SppA (SppABS) 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 SppABS self-processes its own C-termini. We have determined the crystal structure of a proteolytically stable fragment of SppABSK199A 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 Bacillus 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 SppABS competes with a fluorometric peptide substrate for the SppABS 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.
Department of Molecular Biology and Biochemistry, Simon Fraser University , South Science Building, 8888 University Drive, Burnaby, British Columbia, Canada V5A 1S6.
Sung-Eun
SE
Nam
Mark
M
Paetzel
D000595:Amino Acid Sequence; D016282:Aspartic Acid Endopeptidases / Q000737:chemistry*; D001412:Bacillus subtilis / Q000201:enzymology*; D001426:Bacterial Proteins / Q000737:chemistry*; D020134:Catalytic Domain; D017124:Conserved Sequence; D018360:Crystallography, X-Ray; D004795:Enzyme Stability; D006860:Hydrogen Bonding; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D011480:Protease Inhibitors / Q000737:chemistry; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D011499:Protein Processing, Post-Translational; D020836:Protein Structure, Quaternary; D059748:Proteolysis; D013379:Substrate Specificity
D001426:Bacterial Proteins; D010446:Peptide Fragments; D011480:Protease Inhibitors; D016282:Aspartic Acid Endopeptidases; C460085:signal peptide peptidase