Grafting of features of cystatins C or B into the N-terminal region or second binding loop of cystatin A (stefin A) substantially enhances inhibition of cysteine proteinases.

Department of Molecular Biosciences, Section of Veterinary Medical Biochemistry, Swedish University of Agricultural Sciences, Uppsala Biomedical Center, Box 575, SE-751 23 Uppsala, Sweden.
Replacement of the three N-terminal residues preceding the conserved Gly of cystatin A by the corresponding 10-residue long segment of cystatin C increased the affinity of the inhibitor for the major lysosomal cysteine proteinase, cathepsin B, by approximately 15-fold. This tighter binding was predominantly due to a higher overall association rate constant. Characterization of the interaction with an inactive Cys29 to Ala variant of cathepsin B indicated that the higher rate constant was a result of an increased ability of the N-terminal region of the chimeric inhibitor to promote displacement of the cathepsin B occluding loop in the second binding step. The low dissociation rate constant for the binding of cystatin A to cathepsin B was retained by the chimeric inhibitor, which therefore had a higher affinity for this enzyme than any natural cystatin identified so far. In contrast, the N-terminal substitution negligibly affected the ability of cystatin A to inhibit papain. However, substitutions of Gly75 in the second binding loop of cystatin A by Trp or His, making the loop similar to those of cystatins C or B, respectively, increased the affinity for papain by approximately 10-fold. This enhanced affinity was due to both a higher association rate constant and a lower dissociation rate constant. Modeling of complexes between the two variants and papain indicated the possibility of favorable interactions being established between the substituting residues and the enzyme. The second-loop substitutions negligibly affected or moderately reduced the affinity for cathepsin B. Together, these results show that the inhibitory ability of cystatins can be substantially improved by protein engineering.
Mesh Terms:
Amino Acid Sequence, Amino Acid Substitution, Binding Sites, Cathepsin B, Cystatins, Cysteine Endopeptidases, Cysteine Proteinase Inhibitors, Humans, Kinetics, Molecular Sequence Data, Papain, Protein Binding, Recombinant Proteins, Substrate Specificity
Biochemistry Sep. 30, 2003; 42(38);11326-33 [PUBMED:14503883]
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