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 en d of mammalian cystatin B for binding of proteinases was elucidated by muta genesis of the bovine inhibitor. Bovine cystatin B was modeled onto the cry stal structure of the human inhibitor in complex with papain with minimal s tructural changes. Substitution of the two deduced contact residues in the second hairpin loop, Leu-73 and His-75, with Gly resulted in appreciably re duced affinities for papain and cathepsins H and B. These losses indicated that the two residues tonether contribute 20-30% of the free energy of bind ing 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 c athepsin L suggested that the second hairpin loop is less important for inh ibition of this proteinase. Replacement of the contact residue in the C-ter minal end, Tyr-97, with Ala resulted in losses in affinity for papain and c athepsins L and H that were consistent with Tyr-97 contributing 6-12% of th e energy of binding of cystatin B to these enzymes. However, this substitut ion minimally affected the affinity for cathepsin B, indicating that the C- terminal end is of limited importance for binding of this proteinase. All a ffinity decreases were due predominantly to increased dissociation rate con stants. These results show that both the second hairpin loop and the C-term inal end of cystatin B contribute to anchoring: the inhibitor to target pro teinases, each of the two regions interacting with a different domain of th e enzyme. However, the relative contributions of these two interactions var y with the proteinase.