Structural and Functional Analysis of the CspB Protease Required for Clostridium Spore Germination
Figure 5
Dual salt bridges are required for prodomain intramolecular chaperone activity.
(a) Overlay of prodomains from CspB perfringens (teal), Tk-SP (grey), and PCSK9 (pink). (b) PDBe PISA analyses of free energy of prodomain dissociation from mature subtilase, with CspB in teal, PCSK9 in pink, and others in grey. (c) Close-up view of dual salt-bridge interaction at prodomain-subtilase interface. The C-terminus of the prodomain (C, teal) extends toward the substrate-binding pocket. Prodomain Glu35, Glu59 and Arg91 residues are shown in teal; subtilase domain Arg231 and D257 residues are shown in magenta. (d) Analysis of CspB prodomain mutant solubility using Western blotting and Coomassie staining. Cultures expressing cspB variants were induced with IPTG, and aliquots were removed 30 minutes later (“induced-IPTG” sample). Cells were lysed by sonication and centrifuged at high speed; the “cleared lysate” sample represents the soluble fraction. CspB variants were purified by affinity chromatography. Equivalent amounts of samples were resolved by SDS-PAGE and analyzed either by Western blotting using anti-CspB perfringens antisera or by Coomassie staining (bottom gel, affinity-purified CspB).