Crystal Structure of Human Cystatin D, a Cysteine Peptidase Inhibitor with Restricted Inhibition Profile

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-Å resolution, respectively. Human cystatin D presents the typical cystatin fold, with a five-stranded antiparallel ␤-sheet wrapped around a five-turn ␣-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 legumainrelated enzymes.

Checking the conformational stability of cystatin C and its L68Q variant by molecular dynamics studies: Why is the L68Q variant amyloidogenic?

“…Further structural studies of other monomeric cystatins, such as cystatin B in complex with papain (Stubbs et al, 1990), cystatin A (Martin et al, 1995;Staniforth et al, 2001) and cystatin D (Alvarez-Fernandez et al, 2005) have also been performed and suggest a common cystatin fold. This suggestion has been further corroborated on elucidation of the crystal and NMR structures of dimeric wt and L68Q cystatin C (Ekiel et al, 1997;Gerhatz et al, 1998;Janowski et al, 2001).…”

Cystatin M/E Is a High Affinity Inhibitor of Cathepsin V and Cathepsin L by a Reactive Site That Is Distinct from the Legumain-binding Site

“…A three-dimensional model of human cystatin M/E was generated on the basis of the published crystal structure of human cystatin D (30), which revealed that the overall predicted structure of cystatin M/E closely matched that of cystatin D (Fig. 1).…”

The Occluding Loop of Cathepsin B Prevents Its Effective Inhibition by Human Kininogens

“…37 Similarly, HMWK obeys a 2:1 binding stoichiometry, with different rate constants for each binding site. 38 Numerous three-dimensional structures of cystatins and stefins have been described, [39][40][41][42] whereas no structural or mechanistic data are presently available for kininogens.…”