Stucture, function and folding of a novel subtilisin serine protease

• Main Author: Gamble, Michael
• Published: Cardiff University 2010
• Subjects: 572.8
• Online Access: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.584976

ISPs constitute the major cellular proteolytic activity of many bacilli, yet their physiological role, mechanism of regulation and 3D structure were unknown. ISP from B. clausii is expressed as a dimeric inactive precursor. Dimerisation is not involved in regulating ISP activity. The 3D structure of ISP revealed residues from the C-terminus cross over and interact with the adjoining protomer distant from either active site. The mechanism for ISP activation involves inter-molecular processing of the 18 residue N-terminus. The ISP structure exposed a novel mechanism by which proteolytic activity is regulated: The N-terminal extension binds back over the active site with the proline from the conserved LIPY sequence inducing a kink in the polypeptide backbone positioning the hydrolysable peptide bond beyond reach of the catalytic serine. The N-terminal extension acted as a potent inhibitor when added in vitro (Ki of 5 x 10"7 M). The majority of ESPs require a prodomain to fold to a native and active conformation. ISP refolds without a classical prodomain and is therefore thermodynamically stable, in contrast to the kinetically stable ESPs. Removal of calcium from ISP results in loss of activity and protein likely adopting a partially unfolded monomeric state. The structure of ISP indicated that calcium is bound at a high-affinity binding site conserved amongst the subtilases. ISP preferentially degraded unfolded protein rather than substrates in native conformation. Also, ISP has a preference for large hydrophobic residues at the PI and P4 substrate binding sites. This supports the hypothesis that ISP is involved in processing of misfolded/unfolded protein. Molecular insights confirm the primary sequence features novel to the ISPs translate to unique structural, functional, folding and regulatory properties amongst the subtilase family. This allows us to postulate the physiological roles for ISPs and how this role differs from their close relatives, the ESPs.