Elongation on the amino-terminal part of stefin B decreases inhibition of cathepsin H.
Two mutants of the cysteine proteinase inhibitor, stefin B, were prepared by ligating the amino-terminal region from cystatin C and kininogen, members of two other families of cystatin superfamily. The mutant proteins were expressed in Escherichia coli and purified to homogeneity. Inhibition and kinetic constants were determined for authentic and mutated stefins against the four different cysteine proteinases, papain and human cathepsins B, L and H. Inhibition of both amino-terminal elongated stefin B mutants was decreased particularly for cathepsin H. A model of the tertiary structure of cathepsin H and its complex with stefin B was constructed. The framework for the model of cathepsin H consisted of structurally conserved regions from tertiary structures of three cysteine proteinases. Variable regions were selected from fragments of other proteins from the protein data base. We suggest that reduced binding of stefins with elongated amino termini is caused by the mini chain of cathepsin H which is probably in close proximity to the amino termini in the complexes. This mini chain is bridged to Cys214 and has already been proposed to be responsible for the aminopeptidase activity of cathepsin H. We conclude that the amino-terminal region of stefin B plays an important role in determining the strength of inhibition of cathepsin H.
Jerala
R
R
Department of Biochemistry and Molecular Biology, Jozef Stefan Institute, Ljubljana, Slovenija.
Kroon-Zitko
L
L
Popovic
T
T
Turk
V
V
D000595:Amino Acid Sequence; D001483:Base Sequence; D056655:Cathepsin H; D002403:Cathepsins / Q000037:antagonists & inhibitors*; D055313:Cystatin B; D015891:Cystatins / Q000737:chemistry* / Q000235:genetics / Q000494:pharmacology; D003546:Cysteine Endopeptidases; D015853:Cysteine Proteinase Inhibitors / Q000737:chemistry* / Q000235:genetics / Q000494:pharmacology; D004247:DNA; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary
C524994:CSTB protein, human; D015891:Cystatins; D015853:Cysteine Proteinase Inhibitors; D055313:Cystatin B; D004247:DNA; D002403:Cathepsins; D003546:Cysteine Endopeptidases; C534126:CTSH protein, human; D056655:Cathepsin H