In the presence of oligomeric chondroitin 4-sulfate (C4-S), cathepsin K (catK) forms a specific complex that was shown to be the source of the major collagenolytic activity in bone osteoclasts. C4-S forms multiple contacts with amino acid residues on the backside of the catK molecule that help to facilitate complex formation. As cathepsin L does not exhibit a significant collagenase activity in the presence or in the absence of C4-S, we substituted the C4-S interacting residues in catK with those of cathepsin L. Variants revealed altered collagenolytic activities with the largest inhibitory effect shown by the hexavariant M5. None of the variants showed a reduction in their gelatinolytic and peptidolytic activities when compared with wild-type catK, indicating no structural alteration within their active sites. However, the crystal structure of the M5 variant in the presence of oligomeric C4-S revealed a different binding of chondroitin 4-sulfate. C4-S is not continuously ordered as it is in the wildtype catK⅐C4-S complex. The orientation and the direction of the hexasaccharide on the catK surface have changed, so that the hexasaccharide is positioned between two symmetry-related molecules. Only one M5 variant molecule of the dimer that is present in the asymmetric unit interacts with C4-S. These substitutions have changed the mode of catK binding to C4-S and, as a result, have likely affected the collagenolytic potential of the variant. The data presented here support our hypothesis that distinct catK/C4-S interactions are necessary for the collagenolytic activity of the enzyme.Collagen degradation is a natural process observed in bone remodeling, wound healing, and organ development. However, excessive collagen degradation is implicated in many serious diseases such as osteoporosis, different forms of arthritis, and some vascular disorders. Triple-helical collagens are the major organic components of bone matrix (type I collagen) and of cartilage (type II collagen). These collagens are highly resistant to general proteolysis, and it requires specific peptidases for their degradation. Cathepsin K (catK), 7 a member of the papain family of lysosomal cysteine peptidases, is the predominant peptidase of bone-degrading osteoclasts (1, 2). It has the ability, unique among mammalian proteinases, of cleaving triple-helical collagen at multiple sites (3, 4). Analysis of catK activities revealed that the collagen-degrading activity is not proportional to the general peptidase activity suggesting some other factors affect the specificity of catK. It was shown (5, 6) that the ability to cleave collagen is highly dependent on the formation of an oligomeric complex of catK with glycosaminoglycans, in particular, with chondroitin 4-sulfate (C4-S). Glycosaminoglycans are naturally present in bone and cartilage in sufficient quantities to form complexes with catK. Complex formation with glycosaminoglycans is unique for catK among other papain-like proteases (6, 7). It is remarkable that monomeric catK has no significant collag...