Structural and mechanistic insights into collagen degradation by a bacterial collagenolytic serine protease in the subtilisin family.

A number of proteases in the subtilisin family derived from environmental or pathogenic microorganisms have been reported to be collagenolytic serine proteases. However, their collagen degradation mechanisms remain unclear. Here, the degradation mechanism of type I collagen fibres by the S8 collagenolytic protease MCP-01, from Pseudoalteromonas sp. SM9913, was studied. Atomic force microscopy observation and biochemical analysis confirmed that MCP-01 progressively released single fibrils from collagen fibres and released collagen monomers from fibrils mainly by hydrolysing proteoglycans and telopeptides in the collagen fibres. Structural and mutational analyses indicated that an enlarged substrate-binding pocket, mainly composed of loops 7, 9 and 11, is necessary for collagen recognition and that the acidic and aromatic residues on these loops form a negatively charged, hydrophobic environment for collagen binding. MCP-01 displayed a non-strict preference for peptide bonds with Pro or basic residues at the P1 site and/or Gly at the P1' site in collagen. His211 is a key residue for the P1-basic-residue preference of MCP-01. Our study gives structural and mechanistic insights into collagen degradation of the S8 collagenolytic protease, which is helpful in developing therapeutics for diseases with S8 collagenolytic proteases as pathogenic factors and in studying environmental organic nitrogen degradation mechanisms.