Deletion of the carboxy terminal part of stefin B does not have a major effect for binding to papain.
Determination of crystal structures of chicken cystatin and human stefin B complexed with papain revealed a novel model of protease inhibition and also structural differences between two cysteine proteinase inhibitor (CPI) families. According to the 3D alignment, stefins have an extension of 9 amino acids on their carboxy terminus in comparison with cystatins. The extension was not expected to make a major contribution to interaction with the enzyme. A deletion mutant of stefin B, corresponding in length to the carboxy terminal sequence of chicken cystatin, was constructed by the use of polymerase chain reaction (PCR). This (C3S, delta 89-98) human stefin B, 10 amino acids shorter, inhibited papain with a Ki of 0.012 nM which is comparable to the Ki of 0.03 nM for authentic, nondeleted recombinant stefin B. This finding thus confirms the tertiary structure-based alignment.
Jerala
R
R
Department of Biochemistry, Jozef Stefan Institute, Ljubljana, Yugoslavia.
Kroon-Zitko
L
L
Kopitar
M
M
Popovic
T
T
Turk
V
V
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002872:Chromosome Deletion; D055313:Cystatin B; D015891:Cystatins / Q000235:genetics / Q000378:metabolism*; D004247:DNA / Q000235:genetics; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D008969:Molecular Sequence Data; D009154:Mutation; D010206:Papain / Q000378:metabolism*; D013329:Structure-Activity Relationship
C524994:CSTB protein, human; D015891:Cystatins; D055313:Cystatin B; D004247:DNA; D010206:Papain