Mechanism of interaction of the mammalian cysteine protease inhibitors, cystatin A and B, with target proteases

Human cystatin A was shown to bind rapidly and strongly to papain and cathepsin L, with Åj of 0.2-20 pM and £ass of -3-5x10^ whereas the affinities for actinidin and cathepsins B, C and H were weaker (Kj 1-40 nM). The inhibition of cathepsin B was ~100-fold slower than that of papain, i. e. fcass was ~104 M’^-s'1. The binding to papain was consistent with a one-step binding mechanism. An N-terminally truncated cystatin A variant had an appreciably reduced affinity for papain, indicating the importance of this region for interaction with cysteine proteases.Mutations in the second hairpin loop of cystatin B, Leu-73—>Gly and His-75—>Gly, decreased the affinity for papain and cathepsins L, H and B to an extent suggesting that this region contributes 20-30 % of the binding energy of cystatin B to target enzymes. A mutation in the C-terminal end of cystatin B, Tyr-97-»Ala, similarly indicated that this end contributes 6-12 % of the binding energy to papain and cathepsins L and H but is of limited importance for cathepsin B binding. The increased fcjiss for the binding of the mutants to proteases suggests that the two regions are important for complex stability.Human and bovine wild-type cystatin B were shown to have indistinguishable inhibitory properties towards cysteine proteases, binding tightly to the endopeptidases, papain and cathepsin L, and more weakly to the exopeptidases, cathepsins H and B. Mutation of the single Cys residue, Cys-3, in cystatin B showed that this residue is involved in the binding of the inhibitor to target enzymes. Cys-3 is most important for cathepsin B binding and for the bovine inhibitor.Sequential truncation of four residues from the N-terminal end of cystatin B resulted in progressively impaired affinities for papain and cathepsins L, H and B. The highest affinity loss was caused by removal of Cys-3, showing that this residue is most important for the binding. The decreased affinities ofthe truncated cystatin B mutants for papain and cathepsin H were due mainly to an increased Åtfiss» indicating that the N-terminal region keeps the inhibitor anchored to these enzymes in the complexes.