The C4-S chain is not continuously ordered; there are gaps in the electron density between the hexasaccharide terminal residues

The C4-S chain is not continuously ordered; there are gaps in the electron density between the hexasaccharide terminal residues. of oligomeric C4-S revealed Lomeguatrib a different binding of chondroitin 4-sulfate. C4-S is not continuously ordered as it is in the wild-type catKC4-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 Lomeguatrib 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. Keywords:Chondroitin Sulfate, Collagen, Cysteine Protease, Protein Domains, Protein Structure, Protein-Protein Interactions, Proteoglycan Structure, Cathepsin, Collagenase == Introduction == 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),7a 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 collagenase activity (6). Therefore, any disruption of the complex formation between catK and glycosaminoglycans might provide a therapeutic effect in collagen degradation-related diseases. On the other hand, the inhibition of catK by excess concentrations of glycosaminoglycans might be Lomeguatrib a contributing factor to bone anomalies as seen in mucopolysaccharidoses (8). Earlier we had solved the structure of the WT catK:C4-S complex that showed that each hexasaccharide segment of the C4-S binds one catK molecule (9). As a result, an oligomer of 17-kDa C4-S binds 12 catK molecules. The C4-S oligosaccharide runs continuously through the crystal lattice and spans six unit cell lengths of theaaxis (42.0 ). The Lomeguatrib interpretation of WT catKC4-S complex structure did not fully explain the effect that C4-S binding had on the catK collagenolytic activity. The oligosaccharide chain binding site was far from the active site Cys25, and the latter was completely exposed to substrates. There were no changes in the catK structure Mouse monoclonal to CD3E compared with the many structures of catK determined earlier Lomeguatrib in the absence of C4-S. At least 12 amino acid residues on the catK surface were identified as being involved in the C4-S binding. As cathepsin L (catL) does not show a significant collagenase activity neither in the presence nor absence of C4-S (7), we have substituted six C4-S interacting residues that are specific for catK binding by the appropriate residues present in catL. The aim of this study has been to determine whether these residues contribute to the formation of the collagenolytically active C4-ScatK complex. Several variants addressing the individual interactions were generated, and their activities toward triple-helical collagen and noncollageneous substrates were determined. The hexavariant (M5) revealed that it has the strongest effect on the collagenolytic activity (60% inhibition). Interestingly, this variant is still capable of binding C4-S, though in an altered manner. This strongly suggests that the collagenase activity of cathepsin K requires a highly specific interaction with C4-S. Here, we report the structure of the M5 catK variant in complex with the 17-kDa C4-S. == EXPERIMENTAL PROCEDURES == == == == == == Materials == Z-Gly-Pro-Arg-MCA and Z-Leu-Arg-MCA were purchased from Bachem Feinchemikalien, Inc. (Bubendorf, Switzerland). C4-S, DTT, and EDTA were obtained from Sigma. Recombinant WT human catK was expressed inPichia pastorisas described previously (10). == Construction of CatK Variants.