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 1H-15N HSQC NMR titration study with three different tropocollagen analogues ((POG)10)3, ((GPOG)7PRG)3, and (GPRG(POG)7C-carbamidomethyl)3, mapped a saddle-like binding cleft on CBD. NMR titrations with three nitroxide spin-labeled analogues of collagenous peptide, (PROXYL-G(POG)7PRG)3, (PROXYL-G(POG)7)3, and (GPRG(POG)7C-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-ray scattering 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.
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