The role of Gly-4 of human cystatin A (stefin A) in the binding of target proteinases. Characterization by kinetic and equilibrium methods of the interactions of cystatin A Gly-4 mutants with papain, cathepsin B, and cathepsin L.

Department of Veterinary Medical Chemistry, Swedish University of Agricultural Sciences, Uppsala Biomedical Center.
The importance of the evolutionarily conserved Gly-4 residue for the affinity and kinetics of interaction of cystatin A with several cysteine proteinases was assessed by site-directed mutagenesis. Even the smallest replacement, by Ala, resulted in approximately 1000-, approximately 10- and approximately 6000-fold decreased affinities for papain, cathepsin L, and cathepsin B, respectively. Substitution by Ser gave further 3-8-fold reductions in affinity, whereas the largest decreases, >10(5)-fold, were observed for mutations to Arg and Glu. The kinetics of inhibition of papain by the mutants with small side chains, Ala and Ser, were compatible with a one-step bimolecular reaction similar to that with wild-type cystatin A. The decreased affinities of these mutants for papain and cathepsin L were due exclusively to increased dissociation rate constants, but the reduced affinities for cathepsin B were due also to decreased association rate constants. The latter finding indicates that the intact N-terminal region serves as a guide directing cystatin A to the active site of cathepsin B, as has been proposed for cystatin C. The kinetics of binding of the mutants with charged side chains, Arg and Glu, to papain were consistent with a two-step binding mechanism, in which the mutant side chains are accommodated in the complex by a conformational change. The NMR solution structure of the Ala and Trp mutants showed only minor changes compared with wild-type cystatin A, indicating that the large reductions in affinity for proteinases are not due to altered structures of the mutants. Instead, a side chain larger than a hydrogen atom at position 4 affects the interaction with the proteinase most likely by interfering with the binding of the N-terminal region.
Mesh Terms:
Binding, Competitive, Cathepsin B, Cathepsin L, Cathepsins, Circular Dichroism, Crystallography, X-Ray, Cystatin A, Cystatins, Cysteine Endopeptidases, Cysteine Proteinase Inhibitors, Endopeptidases, Glycine, Humans, Kinetics, Magnetic Resonance Spectroscopy, Models, Molecular, Mutagenesis, Insertional, Papain, Protein Binding, Recombinant Proteins
Biochemistry May. 19, 1998; 37(20);7551-60 [PUBMED:9585570]
Download 2 Interactions For This Publication
1673
Switch View:
  • Interactions (2)