Unidirectional Binding of Clostridial Collagenase to Triple Helical Substrates

by Philominathan, Sagaya Theresa Leena; Koide, Takaki; Hamada, Kentaro; Yasui, Hiroyuki; Seifert, Soenke; Matsushita, Osamu; Sakon, Joshua

Histotoxic clostridia produce collagenases responsible for extensive tissue destruction in gas gangrene. The C-terminal collagen-binding domain (CBD) of these enzymes is the minimal segment required to bind to collagen fibril. Collagen binding efficiency of CBD is more pronounced in the presence of Ca2+. We have shown that CBD can be functional to anchor growth factors in local tissue. A H-1-N-15 HSQC NMR titration study with three different tropocollagen analogues ((POG)(10))(3), ((GPOG)(7)PRG)(3), and (GPRG(POG)(7)C-carbamidomethyl)(3), mapped a saddle-like binding cleft on CBD. NMR titrations with three nitroxide spin-labeled analogues of collagenous peptide, (PROXYL-G(POG)(7)PRG)(3), (PROXYL-G(POG)(7))(3), and (GPRG(POG)(7)C-PROXYL)(3) (where PROXYL represents 2,2,5,5-tetramethyl-L-pyrrolidinyloxy), unambiguously demonstrated unidirectional binding of CBD to the tropocollagen analogues. Small angle x-rays cattering data revealed that CBD binds closer to a terminus for each of the five different tropocollagen analogues, which in conjunction with NMR titration studies, implies a binding mode where CBD binds to the C terminus of the triple helix.

Journal
Journal of Biological Chemistry
Volume
284
Issue
16
Year
2009
Start Page
10868-10876
URL
https://dx.doi.org/10.1074/jbc.m807684200
ISBN/ISSN
1083-351X; 0021-9258
DOI
10.1074/jbc.m807684200