The Role of Basic Amino Acid Surface Clusters on the Collagenase Activity of Cathepsin K

Abstract

Cathepsin K is a highly potent collagenase in osteoclasts and responsible for bone degradation. We have previously demonstrated that its unique collagenolytic activity is modulated by glycosaminoglycans that form high molecular complexes with the protease. However, mutational analysis of a specific glycosaminoglycan-cathepsin K binding site only led to a 60% reduction of the collagenolytic activity suggesting additional glycosaminoglycan binding sites or other determinants controlling this activity. We identified 8 cathepsin K specific arginine/lysine residues that form three positively charged clusters at the bottom part of the protease opposing the active site. These residues are highly conserved among mammalian, avian, and reptilian cathepsin K orthologues and to a lesser degree in amphibian and fish specimens. Mutational analysis of these residues revealed an approximately 50% reduction of the collagenolytic activity when the basic amino acids in cluster 2 (K106, K108, R108, R111) were mutated into alanine residues and resulted in a 100% loss of this activity when the mutations were expanded into cluster 3 (K122, R127). Cluster 1 mutations (K77, R79) had no effect. A partial rescue effect was observed when the hexa-mutant variant was combined with three mutations in the previously identified glycosaminoglycan binding site (N190, K101, L195K) indicating the relevance of at least two independent interaction sites. Amino acid substitutions in all sites had no effect on the catalytic efficacy of the protease variants as reflected in their unaltered peptidolytic and gelatinolytic activities and their overall protein stabilities. This study suggests that the basic amino acid clusters in cathepsin K are either involved in alternative glycoasaminoglycan binding sites, play other roles in the formation of collagenolytically active protease complexes or contribute in a yet unknown manner to the specific binding to collagen.

Structural basis of collagen fiber degradation by cathepsin K

“…1 B and C). Interestingly, R108, R111, K122, R127, and K214 are unique to CatK among cysteine cathepsins (21). A third binding site is seen via the interaction of the G2 chain with Q92 of the adjacent molecule thereby identifying a CatK dimer (Fig.…”

“…Two long-stretched GAG binding sites were identified, which include for the G1 GAG molecule the basic amino acid residues K119, K122, R123, and R127 and for the G2 molecule, residues K40, K41, R108, R111, R127, and K214. An analysis of species conserved CatK specific surface arginine and lysine residues demonstrated that R108, R111, K122, and R127 were important for the collagenolytic activity of CatK (21). Interestingly, a study combining modeling, binding kinetics, and crosslinking experiments has also suggested additional GAG binding sites.…”

Collagen Type I as a Ligand for Receptor-Mediated Signaling

“…Since cathepsin K is the only papain-like cysteine protease capable of cleaving triple helical collagen, it is of significant interest as a pharmaceutical target [90,93]. Structural analysis revealed that for cathepsin K to demonstrate its collagenase activity, a dimer has to form an oligomeric complex with a glycosaminoglycan and dock onto a collagen molecule with its central grove [92,94,95]. The presence of glycosaminoglycans allows access to the triple helix, leading to the cutting of the fibril into smaller sub-fibrils and a simultaneous release of glycosaminoglycans.…”

Cell–matrix interactions: focus on proteoglycan–proteinase interplay and pharmacological targeting in cancer

“…Recently, the presence of three positively‐charged clusters has been reported at the bottom part of the protease opposing the active site, and these are involved in alternative GAG binding sites. They may play other roles in the formation of collagenolytically active protease complexes, or contribute in an as yet unknown manner to the specific binding to collagen . Therefore, any disruption of the complex formation between cathepsin K and GAGs might be of therapeutic value in collagen degradation‐related diseases such as cancer.…”