B

Fig. 1 a Schematic representation of the cystatin B gene structure and the nucleotide variations found. Polymorphic variants of cystatin B are shown above the gene. Mutations causing EPM1 are shown below the gene. Numbers preceded by "c" correspond to position in the complementary DNA (cDNA), A of the ATG being 1. The underlined mutations are the first ones described by Pennacchio et al. (1996). b Agarose gel and sequence of PCR-amplified normal alleles containing two or three copies of the dodecamer. The genotype is showing above each lane domain and the first hairpin loop, containing the highly conserved QVVAG motif, with minor contributions from the second hairpin loop. The carb-oxyl terminus of cystatin B is an additional site of interaction, dominated by hydrophobic contacts. Gly4 is close to but not in direct contact with the Cys25 in the active site of papain. Amino-terminal deletions of recombinant chicken cystatin provided functional support to the crystallographic data. In particular, a protein starting at Gly9 (equivalent to Gly4 of human cystatin B) exhibited 5000-fold to 10 000-fold weaker inhibition of papain (Machleidt et al. 1989, 1991). Directed mutagenesis, deletions, and elongations identified regions of cystatin B involved in its biological activity that are consistent with data for chicken cystatin (Abrahamson et al. 1987; Thiele et al. 1990; Jerala et al. 1994; Pol and Bjork 2003).

In vitro, cystatin B is a tight-binding reversible inhibitor of papain and cathepsins B, H, L, and S (Green et al. 1984; Popovic et al. 1988; Bromme et al. 1991). Except for cathepsin B, which is also present at the cytoplasm, the others are lysosomal proteinases responsible for protein degradation (Schwartz and Barrett 1980; Barrett and Kirschke 1981; Bohley and Seglen

Fig. 2 a Ribbon representation of the cystatin B (blue)-papain (gray) complex. b, c Magnification of the active site in the wild type and the G4R mutant cystatin C. The location of Gly4 in the wild-type protein is shown with a magenta sphere, and that of Arg4 with a group of red spheres. This side chain is in steric conflict with the binding site on papain, and is likely to reduce the inhibitory activity of the G4R cystatin B mutant

Fig. 2 a Ribbon representation of the cystatin B (blue)-papain (gray) complex. b, c Magnification of the active site in the wild type and the G4R mutant cystatin C. The location of Gly4 in the wild-type protein is shown with a magenta sphere, and that of Arg4 with a group of red spheres. This side chain is in steric conflict with the binding site on papain, and is likely to reduce the inhibitory activity of the G4R cystatin B mutant

1992). Some cathepsins conduct nonselective protein degradation, while others have tissue- and substrate-specific functions.

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