title: Structure-activity analysis of cathepsin K/chondroitin 4-sulfate interactions.
abstract: 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 wild-type catK·C4-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.
authors.affiliation: Group in Protein Structure and Function, Department of Biochemistry, School of Molecular and Systems Medicine, University of Alberta, Edmonton, Alberta T6G 2H7, Canada.
authors.forename: Maia M
authors.initials: MM
authors.lastname: Cherney
authors.forename: Fabien
authors.initials: F
authors.lastname: Lecaille
authors.forename: Martin
authors.initials: M
authors.lastname: Kienitz
authors.forename: Ferez S
authors.initials: FS
authors.lastname: Nallaseth
authors.forename: Zhenqiang
authors.initials: Z
authors.lastname: Li
authors.forename: Michael N G
authors.initials: MN
authors.lastname: James
authors.forename: Dieter
authors.initials: D
authors.lastname: Brömme
mesh_terms: D019943:Amino Acid Substitution; D056657:Cathepsin K / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002809:Chondroitin Sulfates / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D017364:Collagenases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D018360:Crystallography, X-Ray; D006801:Humans; D020125:Mutation, Missense; D010010:Osteoclasts / Q000201:enzymology*; D011485:Protein Binding; D055503:Protein Multimerization / Q000502:physiology*; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013329:Structure-Activity Relationship
chemical_list: D011994:Recombinant Proteins; D002809:Chondroitin Sulfates; C534130:CTSK protein, human; D056657:Cathepsin K; D017364:Collagenases