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. Significance Fibrillar collagens constitute 90% of the organic bone matrix and are subjected either to physiological remodeling or excessive degradation during diseases such as osteoporosis. Cathepsin K is the critical collagenase in bone and represents a major antiresorptive drug target. Despite its critical role in bone remodeling, its mechanism of collagen degradation remained elusive. Here, we demonstrate that the degradation of fibrillar collagen requires the presence of a cathepsin K dimer bound at the surface of collagen fibers via glycosaminoglycans. Structural modifications of the protease dimerization site or the removal of collagen fiber-associated glycosaminoglycans specifically block fibrillar collagen degradation. The provided structure allows the development of a strategy to inhibit this highly relevant drug target in a substrate-specific manner.

During ageing, changes occur in the collagen network that contribute to various pathological phenotypes in the skeletal, vascular, and pulmonary system. The aim of this study was to investigate the consequences of age-related modifications on the mechanical stability and proteolytic degradation of type I collagen. Analyzing mouse tail and bovine bone collagen, we found that collagen at both fibril and fiber levels varies in rigidity and Young′s modulus through different physiological changes, which correlates with changes in cathepsin K (CatK)-mediated degradation. A decreased susceptibility to CatK-mediated hydrolysis of fibrillar collagen was observed following mineralization and advanced glycation end-product-associated modification. However, ageing of bone increased CatK-mediated osteoclastic resorption by ~27% and negligible resorption was observed when osteoclasts were cultured on mineral-deficient bone. We observed significant differences in the excavations generated by osteoclasts and C-terminal telopeptide release during bone resorption under distinct conditions. Our data indicate that modification of collagen compromises its biomechanical integrity and affects CatK-mediated degradation both in bone and tissue, and thus contribute to our understanding of extracellular matrix ageing.

Excessive cathepsin K (catK)-mediated turnover of fibrillar type I and II collagens in bone and cartilage leads to osteoporosis and osteoarthritis. However, little is known about how catK degrades compact collagen macromolecules. The present study is aimed to explore the structural and mechanical consequences of collagen fiber degradation by catK. Mouse tail type I collagen fibers were incubated with either catK or non-collagenase cathepsins. Methods used include scanning electron microscopy, protein electrophoresis, atomic force microscopy, and tensile strength testing. Our study revealed evidence of proteoglycan network degradation, followed by the progressive disassembly of macroscopic collagen fibers into primary structural elements by catK. Proteolytically released GAGs are involved in the generation of collagenolytically active catK-GAG complexes as shown by AFM. In addition to their structural disintegration, a decrease in the tensile properties of fibers was observed due to the action of catK. The Young's moduli of untreated collagen fibers versus catK-treated fibers in dehydrated conditions were 3.2 ± 0.68 GPa and 1.9 ± 0.65 GPa, respectively. In contrast, cathepsin L, V, B, and S revealed no collagenase activity, except the disruption of proteoglycan-GAG interfibrillar bridges, which slightly decreased the tensile strength of fibers.