Crystallographic analysis of Bacillus subtilis signal peptide peptidase (SppA)

Author: 
Date created: 
2012-12-06
Identifier: 
etd7595
Keywords: 
Signal peptide peptidase A
Self-compartmentalized protease
Ser/Lys protease
Abstract: 

Secreted proteins are initially synthesized in a precursor form that contains an N-terminal signal peptide for targeting the proteins to the cytoplasmic membrane. Upon translocation across the membrane via the Sec machinery, the signal peptide is cleaved off by signal peptidase. The remnant membrane embedded signal peptide is then cleaved within its hydrophobic core by signal peptide peptidase A (SppA). SppAs are membrane bound peptidases found in archaea, plant chloroplasts and bacteria. SppAs utilize a serine nucleophile and a lysine general base as catalytic residues. My PhD research has focused on the Bacillus subtilis SppA (SppABS). During my PhD study, I solved the three-dimensional structure of SppABS using X-ray crystallography, allowing us to study the difference and similarities between Gram-positive SppABS and the previously solved Gram-negative Escherichia coli SppA (SppAEC). Both proteins form similarly sized dome-shaped multi-subunit structures where the catalytic residues reside inside the concave portion of the dome. However, each subunit of SppABS is half the size of the SppAEC subunit, thus SppABS is an octameric complex while SppAEC is a tetramer. The structure revealed eight active sites in SppABS where three of the protomers come together to form one complete active site whereas SppAEC contains only four active sites. Structural analysis on the substrate binding pockets S1 and S3, along with activity assays using a range of peptide substrates, revealed that SppABS prefers substrates with leucine, arginine or tyrosine at the P1 position – a significantly different profile from SppAEC which prefers leucine but not arginine or tyrosine at the P1 position. I also solved the crystal structure of SppABS in complex with its own C-terminus bound within its active site. This structure has provided information on how the enzyme recognizes its substrates, confirms our previous SppABS substrate preference analysis and has posed a new question “Why does SppABS cleave its own C-terminus?” We show that SppABS cleaves its own C-terminus in an intra-complex fashion and mutational analysis shows that Tyr331 is important for self-cleavage. I also showed that SppABS is able to digest folded proteins which suggests that SppABS possibly has a membrane quality control function.

Document type: 
Thesis
Rights: 
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Supervisor(s): 
Mark Paetzel
Department: 
Science: Department of Molecular Biology and Biochemistry
Thesis type: 
(Thesis) Ph.D.
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