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

Summary, in English

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.

• Author: Ingemar Björk; Ingrid Brieditis; Magnus Abrahamson
• Department/s: Division of Clinical Chemistry and Pharmacology
• Publishing year: 1995
• Language: English
• Pages: 513-518
• Publication/Series: Biochemical Journal
• Volume: 306
• Issue: 2
• Links: http://www.pubmedcentral.nih.gov/picrender.fcgi?artid=1136547&blobtype=pdf
• Document type: Journal article
• Publisher: Portland Press
• Topic: Biochemistry and Molecular Biology
• Status: Published
• ISBN/ISSN/Other: ISSN: 0264-6021