Structural basis of collagen fiber degradation by cathepsin K
Structural basis of collagen fiber degradation by cathepsin K
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DOI：
10.1073/pnas.1414126111
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发表时间：
2014-12-09
影响因子：
11.1
通讯作者：
Broemme, Dieter
中科院分区：
文献类型：
--
作者：
Aguda, Adeleke H.;Panwar, Preety;Broemme, Dieter
组织蛋白酶K是骨中主要的胶原溶解蛋白酶，其促进生理性以及病理性骨降解。尽管其在骨重建中的关键作用以及作为用于治疗骨质疏松症的备受追捧的药物靶标，但组织蛋白酶K降解胶原纤维的机制仍然难以捉摸。在这里，我们报告的结构，胶原活性的组织蛋白酶K蛋白二聚体。组织蛋白酶K被组织成细长的C形蛋白酶二聚体，揭示了一个假定的胶原蛋白结合界面辅助糖胺聚糖。胶原结合二聚体的分子建模表明，参与非活性位点的氨基酸残基，Q21和Q92，在胶原展开。这些位点的突变以及二聚体蛋白质-蛋白质界面的扰动完全抑制组织蛋白酶-K介导的纤维降解，而不影响明胶或合成肽的水解。使用扫描电子显微镜，我们证明了组织蛋白酶K在胶原纤维的纤维间隙区域的边缘的特异性结合，这表明在原胶原分子的N-和C-末端的初始裂解事件。胶原纤维降解产物的Edman降解分析揭示了这些初始裂解位点。我们建议，一个组织蛋白酶K分子结合胶原蛋白结合的糖胺聚糖在差距地区，并招募第二个蛋白酶分子，提供了一个三螺旋胶原蛋白的解折叠和切割机制。胶原蛋白相关糖胺聚糖的去除防止组织蛋白酶K结合和随后的纤维水解。组织蛋白酶K二聚体和糖胺聚糖结合位点代表了开发这一重要药物靶点的非活性定点第二代抑制剂的新靶向位点。
Cathepsin K is the major collagenolytic protease in bone that facilitates physiological as well as pathological bone degradation. Despite its key role in bone remodeling and for being a highly sought-after drug target for the treatment of osteoporosis, the mechanism of collagen fiber degradation by cathepsin K remained elusive. Here, we report the structure of a collagenolytically active cathepsin K protein dimer. Cathepsin K is organized into elongated C-shaped protease dimers that reveal a putative collagen-binding interface aided by glycosaminoglycans. Molecular modeling of collagen binding to the dimer indicates the participation of nonactive site amino acid residues, Q21 and Q92, in collagen unfolding. Mutations at these sites as well as perturbation of the dimer protein-protein interface completely inhibit cathepsin-K-mediated fiber degradation without affecting the hydrolysis of gelatin or synthetic peptide. Using scanning electron microscopy, we demonstrate the specific binding of cathepsin K at the edge of the fibrillar gap region of collagen fibers, which suggest initial cleavage events at the N- and C-terminal ends of tropocollagen molecules. Edman degradation analysis of collagen fiber degradation products revealed those initial cleavage sites. We propose that one cathepsin K molecule binds to collagen-bound glycosaminoglycans at the gap region and recruits a second protease molecule that provides an unfolding and cleavage mechanism for triple helical collagen. Removal of collagen-associated glycosaminoglycans prevents cathepsin K binding and subsequently fiber hydrolysis. Cathepsin K dimer and glycosaminoglycan binding sites represent novel targeting sites for the development of nonactive site-directed second-generation inhibitors of this important drug target.