The Importance of the Second Hairpin Loop of Cystatin C for Proteinase Binding. Characterization of the Interaction of Trp-106 Variants of the Inhibitor with Cysteine Proteinases

Abstract

The single Trp of human cystatin C, Trp-106, is located in the second hairpin loop of the proteinase binding surface. Substitution of this residue by Gly markedly altered the spectroscopic changes accompanying papain binding and reduced the affinity for papain, actinidin, and cathepsins B and H by 300-900-fold. The decrease in affinity indicated that 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, corresponding to about 20-30% of the total binding energy. Replacement of Trp-106 by Phe led to a smaller (30-120-fold) decrease in affinity for the four enzymes than Gly substitution. The binding energy of the Phe residue corresponded to 20-45% of that of Trp, showing that a phenyl group can only partly substitute for the indole ring. The reduced affinities of the cystatin C Trp-106 variants for all proteinases studied 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 been formed. This role is partly in contrast to that of the N-terminal region, which increases the affinity of cystatin C for cathepsin B by increasing the association rate constant. Removal of the N-terminal region of the Trp-106-->Gly variant by proteolytic cleavage substantially weakened the binding to papain and cathepsin B. The resulting affinity 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.

Selective Cysteine Protease Inhibition Contributes to Blood-feeding Success of the Tick Ixodes scapularis

“…Now we further show that both I. scapularis salivary cystatins lack the two PW residues in the PIII segment that are instead substituted with a conserved NL dipeptide. Single aa substitutions in the PW dipeptide have been shown to reduce cystatin affinity for cathepsins B and H (19). It is possible that sialostatins L and L2 recruited those two aa substitutions for I. scapularis to get rid of a potentially undesirable or unnecessary inhibitory activity of their salivary cystatins against vertebrate cathepsins B and H, which can diverge these salivary proteins for their target selectivity.…”

Modulation of Human T Cell Responses and Macrophage Functions by Onchocystatin, a Secreted Protein of the Filarial NematodeOnchocerca volvulus

“…Models of the protease-inhibitor interaction suggest that three conserved domains of the cystatins mediate the inhibition of papain-like cysteine proteases (37). The N-terminal conserved inhibitory domain has been shown to have a predominant role because it blocks the active site cleft of the protease (38 -40), whereas the inhibitory potential of the two other domains is dependent on the respective protease (41,42). Given that onchocystatin contains all three conserved domains, it is not surprising that rOv17 strongly inhibits human cysteine proteases like cathepsins S and L. These cathepsins have essential functions in the processing of Ags and in Ag presentation.…”

Crystal Structure of Human Cystatin D, a Cysteine Peptidase Inhibitor with Restricted Inhibition Profile

“…2) Structurally, there is an insertion of 1 amino acid residue in the loop. The isoleucine residue located between Val 37 and Asn 38 deviates the most from the Ala 37 and Ser 38 residues in the corresponding loop segment of CEW cystatin (Ala 35 and Ser 36 in CEW cystatin numbering). This Ile residue is surrounded by the hydrophobic environment provided by the side chains in the C-terminal end of the ␣-helix and those in the end of the fifth ␤-strand.…”

“…The Binding Site for Papain-like Peptidases-The papainbinding site in cystatins is constituted by three well-conserved segments: the flexible N-terminal part, a hairpin loop in the central region (L1), and another toward the C-terminal end (L2) of the sequence (33)(34)(35)(36)(37)(38)(39)(40)(41)(42). These segments form a tripartite wedge-shaped edge (7,8,10,11,43) that enters the catalytic site in a substrate-like manner (10,44).…”