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 Ca(2+). We have shown that CBD can be functional to anchor growth factors in local tissue. A (1)H-(15)N 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-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.
Philominathan
Sagaya Theresa Leena
ST
Department of Chemistry and Biochemistry, University of Arkansas, Fayetteville, Arkansas 72701, USA. pstleena@uark.edu
Koide
Takaki
T
Hamada
Kentaro
K
Yasui
Hiroyuki
H
Seifert
Soenke
S
Matsushita
Osamu
O
Sakon
Joshua
J
D047009:Hathewaya histolytica / Q000201:enzymology*; D003094:Collagen / Q000737:chemistry / Q000378:metabolism; D017364:Collagenases / Q000378:metabolism*; D008958:Models, Molecular; D019906:Nuclear Magnetic Resonance, Biomolecular; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D013113:Spin Labels; D013379:Substrate Specificity
D010455:Peptides; D013113:Spin Labels; D003094:Collagen; D017364:Collagenases