title: Hydrophobic sequences can substitute for the wild-type N-terminal sequence of cystatin A (stefin A) in tight binding to cysteine proteinases selection of high-affinity N-terminal region variants by phage display.
abstract: A phage-display library of the cysteine-proteinase inhibitor, cystatin A, was constructed in which variants with the four N-terminal amino acids randomly mutated were expressed on the surface of filamenteous phage. Screening of this library for binding to papain gave predominantly variants with a glycine residue in position 4. This finding is in agreement with previous conclusions that glycine in this position is essential for tight binding of cystatin A to cysteine proteinases by allowing optimal interaction of the N-terminal region of the inhibitor with the enzyme. In contrast, the first three residues of the variants obtained by the screening were more variable. Two variants were identified with similar affinities for papain as the wild-type inhibitor, but with these residues, Val-Phe-Thr- or Ile-Leu-Leu, differing appreciably from those of the wild-type, Met-Ile-Pro. Other sequences of the N-terminal region, presumably mainly hydrophobic, can thus substitute for the wild-type sequence and contribute similar energy to the inhibitor-proteinase interaction. The two variants binding tightly to papain differed in their affinity for cathepsin B, demonstrating that cystatin variants with increased selectivity for a particular target cysteine proteinase can be obtained by phage-display technology.
authors.affiliation: Department of Veterinary Medical Chemistry, Swedish University of Agricultural Sciences, Uppsala Biomedical Center, Sweden.
authors.forename: K
authors.initials: K
authors.lastname: Ylinenjärvi
authors.forename: M
authors.initials: M
authors.lastname: Widersten
authors.forename: I
authors.initials: I
authors.lastname: Björk
mesh_terms: D000595:Amino Acid Sequence; D001435:Bacteriophages / Q000235:genetics; D001483:Base Sequence; D015891:Cystatins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D017931:DNA Primers; D007700:Kinetics; D011485:Protein Binding
chemical_list: D015891:Cystatins; D017931:DNA Primers; D003546:Cysteine Endopeptidases