THE IMPORTANCE OF THE 2ND HAIRPIN LOOP OF CYSTATIN-C FOR PROTEINASE BINDING, CHARACTERIZATION OF THE INTERACTION OF TRP-106 VARIANTS OF THEINHIBITOR WITH CYSTEINE PROTEINASES

The single Trp of human cystatin C, Trp-106, is located in the second hairpin loop of the proteinase binding surface. Substitution of this r esidue by Gly markedly altered the spectroscopic changes accompanying papain binding and reduced the affinity for papain, actinidin, and cat hepsins B and H by 300-900-fold. The decrease in affinity indicated th at the side chain of Trp-106 contributes a similar free energy, -14 to -17 kJ . mol(-1), to the binding to all four cysteine proteinases, co rresponding to about 20-30% of the total binding energy, Replacement o f Trp-106 by Phe led to a smaller (30-120-fold) decrease in affinity f or the four enzymes than Gly substitution, The binding energy of the P he residue corresponded to 20-45% of that of Trp, showing that a pheny l group can only partly substitute for the indole ring. The reduced af finities of the cystatin C Trp-106 variants for all proteinases studie d were due almost exclusively to increased dissociation rate constants , The second hairpin loop thus contributes to the binding primarily by keeping cystatin C anchored to the proteinase once the complex has be en formed, This role is partly in contrast to that of the N-terminal r egion, which increases the affinity of cystatin C for cathepsin B by i ncreasing the association rate constant. Removal of the N-terminal reg ion of the Trp-106-->Gly variant by proteolytic cleavage substantially weakened the binding to papain and cathepsin B, The resulting affinit y indicated that the first hairpin loop (the ''QVVAG-region''), which is the only region of the proteinase binding surface remaining intact in the truncated variant, contributes 40-60% of the total free energy of binding of cystatin C to both proteinases.