期刊
PROTEIN SCIENCE
卷 21, 期 10, 页码 1554-1565出版社
WILEY-BLACKWELL
DOI: 10.1002/pro.2145
关键词
collagen; binding; NMR; triple-helix; undertwisted collagen
资金
- National Institutes of Health Center for Protein Structure and Function [NCRR COBRE 1 P20RR15569, INBRE P20RR16460]
- AR Biosciences Institute
- Japan Society for the Promotion of Science
- Kagawa University Project Research Fund
- MEXT, Japan [19390032]
- E.I. DuPont de Nemours Co.
- Dow Chemical Company
- Northwestern University
- U.S. DOE [DE-AC02-06CH11357]
- Grants-in-Aid for Scientific Research [23590516, 19390032] Funding Source: KAKEN
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)xPOA(POG)y]3, where x + y = 9 and x > 3). 1H15N heteronuclear single quantum coherence nuclear magnetic resonance (HSQC NMR) titration studies with 15N-labeled CBD demonstrated that the minicollagen binds to a 10 angstrom wide 25 angstrom 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.
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