Unidirectional binding of clostridial collagenase to triple helical substrates

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.