The refined 2.15 A X-ray crystal structure of human liver cathepsin B: the structural basis for its specificity

EMBO J. 1991 Sep;10(9):2321-30. doi: 10.1002/j.1460-2075.1991.tb07771.x.


From the lysosomal cysteine proteinase cathepsin B, isolated from human liver in its two-chain form, monoclinic crystals were obtained which contain two molecules per asymmetric unit. The molecular structure was solved by a combination of Patterson search and heavy atom replacement methods (simultaneously with rat cathepsin B) and refined to a crystallographic R value of 0.164 using X-ray data to 2.15 A resolution. The overall folding pattern of cathepsin B and the arrangement of the active site residues are similar to the related cysteine proteinases papain, actinidin and calotropin DI. 166 alpha-carbon atoms out of 248 defined cathepsin B residues are topologically equivalent (with an r.m.s. deviation of 1.04 A) with alpha-carbon atoms of papain. However, several large insertion loops are accommodated on the molecular surface and modify its properties. The disulphide connectivities recently determined for bovine cathepsin B by chemical means were shown to be correct. Some of the primed subsites are occluded by a novel insertion loop, which seems to favour binding of peptide substrates with two residues carboxy-terminal to the scissile peptide bond; two histidine residues (His110 and His111) in this "occluding loop' provide positively charged anchors for the C-terminal carboxylate group of such polypeptide substrates. These structural features explain the well-known dipeptidyl carboxypeptidase activity of cathepsin B. The other subsites adjacent to the reactive site Cys29 are relatively similar to papain; Glu245 in the S2 subsite favours basic P2-side chains. The above mentioned histidine residues, but also the buried Glu171 might represent the group with a pKa of approximately 5.5 near the active site, which governs endo- and exopeptidase activity. The "occluding loop' does not allow cystatin-like protein inhibitors to bind to cathepsin B as they do to papain, consistent with the reduced affinity of these protein inhibitors for cathepsin B compared with the related plant enzymes.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Binding Sites
  • Cathepsin B / antagonists & inhibitors
  • Cathepsin B / chemistry*
  • Cathepsin B / metabolism
  • Crystallization
  • Cysteine Endopeptidases / chemistry
  • Humans
  • Liver / enzymology*
  • Molecular Sequence Data
  • Papain / chemistry
  • Peptides / metabolism
  • Protein Conformation
  • Sequence Homology, Nucleic Acid
  • Substrate Specificity
  • X-Ray Diffraction


  • Peptides
  • Cysteine Endopeptidases
  • Cathepsin B
  • actinidain
  • Papain