Bacterial collagen-binding domain targets undertwisted regions of collagen.
Clostridium histolyticum collagenase causes extensive degradation of collagen in connective tissue that results in gas gangrene. The C-terminal collagen-binding domain (CBD) of these enzymes is the minimal segment required to bind to a collagen fibril. CBD binds unidirectionally to the undertwisted C-terminus of triple helical collagen. Here, we examine whether CBD could also target undertwisted regions even in the middle of the triple helix. Collageneous peptides with an additional undertwisted region were synthesized by introducing a Gly → Ala substitution [(POG)(x) POA(POG)(y)]₃, where x + y = 9 and x > 3). ¹H-¹⁵N heteronuclear single quantum coherence nuclear magnetic resonance (HSQC NMR) titration studies with ¹⁵N-labeled CBD demonstrated that the minicollagen binds to a 10 Å wide 25 Å long cleft. Six collagenous peptides each labeled with a nitroxide radical were then titrated with ¹⁵N-labeled CBD. CBD binds to either the Gly → Ala substitution site or to the C-terminus of each minicollagen. Small-angle X-ray scattering measurements revealed that CBD prefers to bind the Gly → Ala site to the C-terminus. The HSQC NMR spectra of ¹⁵N-labeled minicollagen and minicollagen with undertwisted regions were unaffected by the titration of unlabeled CBD. The results imply that CBD binds to the undertwisted region of the minicollagen but does not actively unwind the triple helix.
Department of Chemistry and Biochemistry, University of Arkansas, Fayetteville, Arkansas 72701, USA. pstleena@gmail.com
Sagaya Theresa Leena
ST
Philominathan
Takaki
T
Koide
Osamu
O
Matsushita
Joshua
J
Sakon
D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism; D001665:Binding Sites; D003094:Collagen / Q000737:chemistry* / Q000378:metabolism; D017364:Collagenases / Q000737:chemistry* / Q000378:metabolism; D009587:Nitrogen Isotopes; D019906:Nuclear Magnetic Resonance, Biomolecular; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D053838:Scattering, Small Angle; D014961:X-Ray Diffraction
D001426:Bacterial Proteins; D009587:Nitrogen Isotopes; D010455:Peptides; D003094:Collagen; C118796:ColH protein, Clostridium histolyticum; D017364:Collagenases