Probing the functional role of the N-terminal region of cystatins by equilibrium and kinetic studies of the binding of Gly-11 variants of recombinant human cystatin C to target proteinases

Abstract

The interaction between cystatin C variants, in which the evolutionarily conserved Gly-11 residue was substituted by Ala, Glu or Trp, and the cysteine proteinases, papain, ficin, actinidin and cathepsin B, was characterized. The substitutions reduced the affinity of binding in a manner consistent with the Gly residue of the wild-type inhibitor, allowing the N-terminal region to adopt a conformation that was optimal for interaction with target proteinases. Replacement of Gly-11 by Ala resulted in only a 5- to 100-fold reduction in binding affinity. Comparison with the affinities of wild-type cystatin C lacking the N-terminal region indicated that even this small structural change affects the conformation of this region sufficiently to largely abolish its interaction with the weakly binding proteinases, actinidin and cathepsin B. However, the substitution allows interactions of appreciable strength between the N-terminal region and the tightly binding enzymes, papain or ficin. Replacement of Gly-11 with the larger Glu and Trp residues substantially decreased the affinity of binding to all enzymes, from 10(3)- to 10(5)-fold. These substitutions further affect the conformation of the N-terminal region, so that interactions of this region with papain and ficin are also essentially eliminated. The decreased affinities of the three cystatin C variants for papain, ficin and actinidin were due exclusively to increased dissociation rate constants. In contrast, the decreased affinity between cathepsin B and the Ala-11 variant, the only one for which rate constants could be determined with this enzyme, was due almost entirely to a decreased association rate constant. This behaviour is analogous to that observed for forms of cystatin C lacking the N-terminal region and supports the conclusion that the mode of interaction of this region with target proteinases varies with the enzyme as a result of structural differences in the active-site region of the latter.

Parasite-Specific Immunomodulatory Functions of Filarial Cystatin

“…Our K i value determinations of nematode cystatins performed with the human cathepsins B, L, and S may reflect differences in the amino acid sequences. Differences in the N-terminal active domain of cystatins have been shown to change their inhibitory capacity (4,13,14). While the K i values of O. volvulus cystatin and C. elegans cystatin for cathepsins L and S were in the same range, cathepsin B was significantly better inhibited by C. elegans cystatins than by O. volvulus cystatin.…”

Cystatins as Calpain Inhibitors: Engineered Chicken Cystatin- and Stefin B-Kininogen Domain 2 Hybrids Support a Cystatin-Like Mode of Interaction with the Catalytic Subunit of µ-Calpain

“…The fact that the recombinant chimeric calpain inhibitors, but not inhibitorily active KD2 (obtained by limited proteolysis of L-kininogen), are cleaved by calpain at the corresponding peptide bond allows three conclusions: (i) the cystatin-type calpain inhibitors interact with the active site of the catalytic subunit of calpain; (ii) their mode of interaction with calpain is similar to that of cystatins with papain-like cysteine proteinases; (iii) in contrast to natural KD2, the chimeric inhibitors fit imperfectly to the active site of calpain and are therefore slowly cleaved in a substrate-like manner. Attempts to generate permanent calpain inhibitors by modification of the cleavage site do not seem promising because mutations in the corresponding region of cystatin C have been shown to decrease its binding affinity for various cysteine proteinases (Björk et al, 1995).…”

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

“…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).…”