Crystallization of Bacillus subtilis Type I Signal Peptidase S (SipS)

Gram-positive Bacillus subtilis (B. subtilis) signal peptidase I (SPase I) is a membrane-bound endopeptidase that cleaves off the amino-terminal signal peptide from pre-proteins before or after their translocation across the cytoplasmic membrane. B. subtilis has five chromosomal SPases I; SipS, SipT, SipU, SipV, SipW, and two plasmid encoded paralogous SipP. SipS is one of major SPases I in the species which is essential for cell viability. It is also one of the closest of the B. subtilis SPase I enzymes in sequence to the well characterized Gram-negative E. coli SPase I. As a result, SipS was chosen for this research study. B. subtilis SipS uses Ser/Lys catalytic dyad for catalytic activity, utilizing Ser43 and Lys83 in the enzyme. The constructs - SipS Full-Length (FL), SipS ∆2-35 Wild-Type (WT), SipS ∆2-35 S43A and SipS ∆2-35 K83A – were expressed, purified, and screened for crystallization conditions. Catalytically active SipS ∆2-35 WT formed needle shaped crystal clusters whereas SipS ∆2-35 K83A produced initial hits in crystallization conditions containing lithium sulfate. Preliminary data for the catalytic activity of B. subtilis SipS ∆2-35 WT shows that hexaaminecobalt (III) chloride inhibits the enzyme.