Structure-activity Analysis of Cathepsin K/chondroitin 4-sulfate Interactions

J Biol Chem. 2011 Mar 18;286(11):8988-98. doi: 10.1074/jbc.M110.126706. Epub 2010 Dec 30.

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Substitution
  • Cathepsin K / chemistry*
  • Cathepsin K / genetics
  • Cathepsin K / metabolism
  • Chondroitin Sulfates / chemistry*
  • Chondroitin Sulfates / genetics
  • Chondroitin Sulfates / metabolism
  • Collagenases / chemistry*
  • Collagenases / genetics
  • Collagenases / metabolism
  • Crystallography, X-Ray
  • Humans
  • Mutation, Missense
  • Osteoclasts / enzymology*
  • Protein Binding
  • Protein Multimerization / physiology*
  • Protein Structure, Quaternary
  • Protein Structure, Tertiary
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism
  • Structure-Activity Relationship

Substances

  • Recombinant Proteins
  • Chondroitin Sulfates
  • CTSK protein, human
  • Cathepsin K
  • Collagenases

Associated data

  • PDB/3H7D