Substrate recognition by the collagen-binding domain of Clostridium histolyticum class I collagenase
Substrate recognition by the collagen-binding domain of Clostridium histolyticum class I collagenase
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DOI：
10.1074/jbc.m003450200
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发表时间：
2001-03-23
影响因子：
4.8
通讯作者：
Okabe, A
中科院分区：
文献类型：
--
作者：
Matsushita, O;Koide, T;Okabe, A
溶组织梭菌I型胶原酶（ColG）具有节段结构，S1+S2+ S3 a + S3 b。S3 a和S3 b与不溶性胶原蛋白结合，但S2不结合，因此表明S3形成胶原蛋白结合结构域（CBD）。由于S3 a + S3 b显示出最有效的结合底物，这两个结构域的合作结合建议的酶。单体（S3 b）和串联（S3 a + S3 b）CBD结合于仅含有胶原区域的去端胶原。然而，它们不与固定在琼脂糖凝胶珠上的端肽结合。这些结果表明CBD的结合位点存在于胶原区域中。CBD结合到固定的胶原肽，（Pro-Hyp-Gly）（n）和（Pro-Pro-Gly）（n），只有当n大到足以允许肽具有三螺旋构象。它们不与具有相似氨基酸序列的各种肽或缺乏三螺旋构象的明胶结合。CBD不与固定的Glc-Gal二糖结合，所述固定的Glc-Gal二糖附着于胶原区域中的羟基赖氨酸残基的侧链。这些观察结果表明，CBD特异性地识别由胶原区域中的三条多肽链形成的三螺旋构象。
Clostridium histolyticum type I collagenase (ColG) has a segmental structure, S1+S2+S3a+S3b. S3a and S3b bound to insoluble collagen, but S2 did not, thus indicating that S3 forms a collagen-binding domain (CBD). Because S3a+S3b showed the most efficient binding to substrate, cooperative binding by both domains was suggested for the enzyme. Monomeric (S3b) and tandem (S3a+S3b) CBDs bound to atelocollagen, which contains only the collagenous region. However, they did not bind to telopeptides immobilized on Sepharose beads. These results suggested that the binding site(s) for the CBD is(are) present in the collagenous region. The CBD bound to immobilized collagenous peptides, (Pro-Hyp-Gly)(n) and (Pro-Pro-Gly)(n), only when n is large enough to allow the peptides to have a triple-helical conformation. They did not bind to various peptides with similar amino acid sequences or to gelatin, which lacks a triple-helical conformation, The CBD did not bind to immobilized Glc-Gal disaccharide, which is attached to the side chains of hydroxylysine residues in the collagenous region. These observations suggested that the CBD specifically recognizes the triple helical conformation made by three polypeptide chains in the collagenous region.