Abstract
Cystatins are natural inhibitors of papain-like (family C1) and legumain-related (family C13) cysteine peptidases. Cystatin D is a type 2 cystatin, a secreted inhibitor found in human saliva and tear fluid. Compared with its homologues, cystatin D presents an unusual inhibition profile with a preferential inhibition cathepsin S > cathepsin H > cathepsin L and no inhibition of cathepsin B or pig legumain. To elucidate the structural reasons for this specificity, we have crystallized recombinant human Arg(26)-cystatin D and solved its structures at room temperature and at cryo conditions to 2.5- and 1.8-A resolution, respectively. Human cystatin D presents the typical cystatin fold, with a five-stranded anti-parallel beta-sheet wrapped around a five-turn alpha-helix. The structures reveal differences in the peptidase-interacting regions when compared with other cystatins, providing plausible explanations for the restricted inhibitory specificity of cystatin D for some papain-like peptidases and its lack of reactivity toward legumain-related enzymes.
Highlights
Cystatins are natural inhibitors of papain-like and legumain-related cysteine peptidases
Assuming that Asn39 in other type 2 cystatins is directly involved in legumain inhibition, we examined and compared the structures in the back-side loop (BSL) of cystatin D and Chicken egg-white (CEW) cystatin (Fig. 4), the latter of which is a tight binding inhibitor of pig legumain [48]
We have determined the structure of recombinant human cystatin D by x-ray crystallography under both room temperature and cryo conditions (2.5 and 1.8 Å resolution, respectively)
Summary
Of cystatin D for human family C1 peptidases is clearly different from that of cystatin C [1, 16]. It was recently reported that some type 2 cystatins can inhibit mammalian legumain, a cysteine peptidase of family C13, through a novel reactive site located on the side opposite to the papain-binding site [18], in a loop referred to as the back-side loop (BSL). This site results in tight reversible inhibition of pig legumain and is active on human cystatins C, E/M, and F, but not on cystatins A, B, and D. We have crystallized and determined the three-dimensional structure of recombinant human cystatin D, with the aim of clarifying the structural reasons for its selectivity at target enzyme inhibition
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