Nuffield Department of Orthopaedics, Rheumatology and Musculoskeletal Sciences, Kennedy Institute of Rheumatology, University of Oxford, London W6 8LH, United Kingdom.
Proc Natl Acad Sci U S A. 2012 Jul 31;109(31):12461-6. doi: 10.1073/pnas.1204991109. Epub 2012 Jul 3.
Collagenases of the matrix metalloproteinase (MMP) family play major roles in morphogenesis, tissue repair, and human diseases, but how they recognize and cleave the collagen triple helix is not fully understood. Here, we report temperature-dependent binding of a catalytically inactive MMP-1 mutant (E200A) to collagen through the cooperative action of its catalytic and hemopexin domains. Contact between the two molecules was mapped by screening the Collagen Toolkit peptide library and by hydrogen/deuterium exchange. The crystal structure of MMP-1(E200A) bound to a triple-helical collagen peptide revealed extensive interactions of the 115-Å-long triple helix with both MMP-1 domains. An exosite in the hemopexin domain, which binds the leucine 10 residues C-terminal to the scissile bond, is critical for collagenolysis and represents a unique target for inhibitor development. The scissile bond is not correctly positioned for hydrolysis in the crystallized complex. A productive binding mode is readily modeled, without altering the MMP-1 structure or the exosite interactions, by axial rotation of the collagen homotrimer. Interdomain flexing of the enzyme and a localized excursion of the collagen chain closest to the active site, facilitated by thermal loosening of the substrate, may lead to the first transition state of collagenolysis.
基质金属蛋白酶（MMP）家族的胶原酶在形态发生、组织修复和人类疾病中发挥着重要作用，但它们如何识别和切割胶原三螺旋尚未完全了解。在这里，我们报告了一种催化失活的 MMP-1 突变体（E200A）通过其催化和血纤蛋白原结构域的协同作用与胶原的温度依赖性结合。通过筛选 Collagen Toolkit 肽文库和氢/氘交换来映射两个分子之间的接触。MMP-1(E200A)与三螺旋胶原肽结合的晶体结构揭示了长达 115Å 的三螺旋与 MMP-1 两个结构域的广泛相互作用。血纤蛋白原结构域中的一个外位点与切割键 C 末端的亮氨酸 10 残基结合，对于胶原酶解至关重要，并且代表了抑制剂开发的独特靶标。在结晶复合物中，切割键未正确定位用于水解。通过胶原同源三聚体的轴向旋转，很容易模拟出一种有生产力的结合模式，而不会改变 MMP-1 结构或外位点相互作用。酶的结构域弯曲和最接近活性位点的胶原链的局部偏移，通过底物的热松弛来促进，可能导致胶原酶解的第一个过渡态。
