Two mutants of the cysteine proteinase inhibitor, stefin B, were prepared by ligating the aminoterminal 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.Cells of animals and plants contain a variety of cysteine proteinases (CP) similar to papain [I]. Mammalian cysteine proteinases include the lysosomal enzymes cathepsins B, H, L, S and C. These enzymes differ from each other in many respects, such as their pH optima or substrate specificity [2]. Some of them are true endopeptidases, while others display exclusively or additional aminopeptidase (cathepsin H) 13, 41 or carboxypeptidase activity (cathepsin B) [2]. The involvement of CPs in different physiological processes is variable as well. Some cathepsins degrade primarily proteins of the cells themselves in the process of aulophagy, while others take part in the degradation of foreign proteins in processes such as antigen processing and presentation [S -71.The inhibition of cysteine proteinases by protein inhibitors is an important mechanism for regulation of their activity [8]. The number of different cystatins (protein inhibitors which inhibit cysteine proteinases) exceeds the number of target enzymes [9]. The superfamily of cystatins of mammalian origin is usually divided into three families ; stefins, cystatins and kininogens [9, 101. In other organisms, there are more inhibitors which can be classified into those three families or form new families with intermediate properties [11]. Up to now, several cystatins have been isolated from human Knowledge on the mechanism of inhibition of cystatins is based on the crystallographic studies, where the tertiary structures of chicken cystatin [21] and human stefin B [22] were determined as representatives of two families. The mode of interaction with cysteine proteinases was first predicted from computer docking 1211 which was later confirme...
Trifluoroethanol (TFE) has been used to probe differences in the stability of the native state and in the folding pathways of the homologous cysteine protein inhibitors, human stefin A and B. After complete unfolding in 4.5 mol/L GuHCl, stefin A refolded in 11% (vol/vol) TFE, 0.75 mol/L GuHCl, at pH 6.0 and 20 degrees C, with almost identical first-order rate constants of 4.1 s-1 and 5.5 s-1 for acquisition of the CD signal at 230 and 280 nm, respectively, rates that were markedly greater than the value of 0.11 s-1 observed by the same two probes when TFE was absent. The acceleration of the rates of refolding, monitored by tyrosine fluorescence, was maximal at 10% (vol/vol) TFE. Similar rates of refolding (6.2s-1 and 7.2 s-1 for ellipticity at 230 and 280 nm, respectively) were observed for stefin A denatured in 66% (vol/vol) TFE, pH 3.3, when refolding to the same final conditions. After complete unfolding in 3.45 mol/L GuHCl, stefin B refolded in 7% (vol/vol) TFE, 0.57 mol/L GuHCl, at pH 6.0 and 20 degrees C, with a rate constant for the change in ellipticity at 280 nm of 32.8 s-1; this rate was only twice that observed when TFE was absent. As a major point of distinction from stefin A, the refolding of stefin B in the presence of TFE showed an overshoot in the ellipticity at 230 nm to a value 10% greater than that in the native protein; this signal relaxed slowly (0.01 s-1) to the final native value, with little concomitant change in the near-ultraviolet CD signal; the majority of this changes in two faster phases. After denaturation in 42% (vol/vol) TFE, pH 3.3, the kinetics of refolding to the same final conditions exhibited the same rate-limiting step (0.01 s-1) but were faster initially. The results show that similarly to stefin A, stefin B forms its hydrophobic core and predominant part of the tertiary structure faster in the presence of TFE. The results imply that the alpha-helical intermediate of stefin B is highly structured. Proteins 1999;36:205-216.
Stefin A is a member of the cystatin superfamily of proteins which are tight and reversibly binding inhibitors of the papain-like cysteine proteinases. The 'H-NMR and "N-NMR resonances of human stefin A have been sequentially assigned using two-dimensional homonuclear and heteronuclear NMR techniques in conjunction with three-dimensional heteronuclear methods. Characteristic sequential and medium range NOE contacts, J constants and hydrogen exchange data have been used to identify the secondary structural elements of the protein which consists of five anti-parallel P-strands and a single a-helix. There is much similarity between the secondary structural features of stefin A and the homologous protein stefin B in its complex with papain [Stubbs, M. T., Laber, B., Bode, W., Huber, R., Jerala, R., LenarZiE, B. & Turk, V. (1990) EMBO. J. 9, 1939-19471 but also some important differences in regions which are fundamental to the binding event. The principal difference is the presence of two conformationally unrestricted regions in stefin A that form two of the components of the tripartite wedge which docks into the active site of the target proteinase. Specifically, these regions are the five N-terminal residues and the second binding loop, which form a turn and a short helix respectively, in the bound conformation of stefin B.Human stefin A is a member of the cystatin superfamily of proteins, which are tight and reversibly binding inhibitors of the papain-like cysteine proteinases. These inhibitors are believed to help protect cells from inappropriate endogenous or external proteolysis, and are involved in the control mechanism responsible for protein breakdown (Turk and Bode, 1991). The cystatin superfamily has been subdivided into three families on the basis of sequence identity, the number of disulphide bonds present, and the molecular mass of the protein (Barrett et al., 1986). Recently determined protein sequences indicate that the classification could be extended to include additional families (e.g. Kondo et al., 1991). Stefin A is a member of family I. This family (also known as the stefins) consists of single-chain proteins with a molecular mass of approximately 11 kDa, which lack disulphide bonds and carbohydrates. Members of family I1 (the cystatins) have a molecular mass of approximately 13 kDa and are characterised by the presence of two disulphide bonds, which are located towards the C-terminus, and a lack of carbohydrates. Family 111 (the kininogens) comprises plasma proteins which are of a larger molecular size (60-120 kDa) than members Correspondence to J. P.
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