Importance of the second binding loop and the C-terminal end of cystatin B (stefin B) for inhibition of cysteine proteinases.
The importance of residues in the second hairpin loop and the C-terminal end of mammalian cystatin B for binding of proteinases was elucidated by mutagenesis of the bovine inhibitor. Bovine cystatin B was modeled onto the crystal structure of the human inhibitor in complex with papain with minimal structural changes. Substitution of the two deduced contact residues in the second hairpin loop, Leu-73 and His-75, with Gly resulted in appreciably reduced affinities for papain and cathepsins H and B. These losses indicated that the two residues together contribute 20-30% of the free energy of binding of cystatin B to these enzymes and that Leu-73 is responsible for most of this contribution. In contrast, the small decrease in the affinity for cathepsin L suggested that the second hairpin loop is less important for inhibition of this proteinase. Replacement of the contact residue in the C-terminal end, Tyr-97, with Ala resulted in losses in affinity for papain and cathepsins L and H that were consistent with Tyr-97 contributing 6-12% of the energy of binding of cystatin B to these enzymes. However, this substitution minimally affected the affinity for cathepsin B, indicating that the C-terminal end is of limited importance for binding of this proteinase. All affinity decreases were due predominantly to increased dissociation rate constants. These results show that both the second hairpin loop and the C-terminal end of cystatin B contribute to anchoring the inhibitor to target proteinases, each of the two regions interacting with a different domain of the enzyme. However, the relative contributions of these two interactions vary with the proteinase.
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Department of Veterinary Medical Chemistry, Swedish University of Agricultural Sciences, Uppsala Biomedical Center, Box 575, SE-751 23 Uppsala, Sweden.
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D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002417:Cattle; D055313:Cystatin B; D015891:Cystatins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D003546:Cysteine Endopeptidases / Q000737:chemistry / Q000378:metabolism; D015853:Cysteine Proteinase Inhibitors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D018076:DNA, Complementary / Q000302:isolation & purification; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding / Q000235:genetics; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D013050:Spectrometry, Fluorescence; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
C524994:CSTB protein, human; D015891:Cystatins; D015853:Cysteine Proteinase Inhibitors; D018076:DNA, Complementary; D010446:Peptide Fragments; D011994:Recombinant Proteins; D055313:Cystatin B; D003546:Cysteine Endopeptidases