The refined 2.4 A X-ray crystal structure of recombinant human stefin B in complex with the cysteine proteinase papain: a novel type of proteinase inhibitor interaction.

Abstract

A stoichiometric complex of human stefin B and carboxymethylated papain has been crystallized in a trigonal crystal form. Data to 2.37 A resolution were collected using the area detector diffractometer FAST. The crystal structure of the complex has been solved by Patterson search techniques using papain as search model. Starting from the structure of chicken cystatin, the stefin structure was elucidated through cycles of model building and crystallographic refinement. The current crystallographic R factor is 0.19. Like cystatin, the stefin molecule consists of a five stranded beta‐sheet wrapped around a five turn alpha‐helix, but with an additional carboxy terminal strand running along the convex side of the sheet. Topological equivalence of stefin and cystatin reveal the previous sequence alignment to be incorrect in part, through deletion of the intermediate helix. The conserved residues form a tripartite wedge, which slots into the papain active site as proposed through consideration of the tertiary structures of the individual components (Bode et al., 1988). The main interactions are provided by the amino terminal ‘trunk’ (occupying the ‘unprimed’ subsites of the enzyme), and by the first hairpin loop, containing the highly conserved QVVAG sequence, with minor contributions from the second hairpin loop. The carboxyl terminus of stefin provides an additional interaction region with respect to cystatin. The interaction is dominated by hydrophobic contacts. Inhibition by the cysteine proteinase inhibitors is fundamentally different to that observed for the serine proteinase inhibitors.

Expression of a barley cystatin gene in maize enhances resistance against phytophagous mites by altering their cysteine-proteases

“…The cystatin inhibitory mechanism is produced by a tight and reversible interaction with their target enzymes. It involves a tripartite wedge formed by the partially flexible N-terminus containing a glycine residue and two hairpin loops carrying a conserved QxVxG motif and a tryptophan residue, respectively (Stubbs et al 1990). In addition to these three motifs common to all cystatins, PhyCys contain a particular consensus sequence HYFQ]-N) conforming a predicted secondary a-helix structure (Margis et al 1998).…”

Cystatins as Calpain Inhibitors: Engineered Chicken Cystatin- and Stefin B-Kininogen Domain 2 Hybrids Support a Cystatin-Like Mode of Interaction with the Catalytic Subunit of µ-Calpain

“…In the present work, we investigate the mechanism of temporary inhibition of µ-calpain by chicken cystatin-KD2 hybrids and the potential reasons for it. The second disulphide loop (Cys95-Cys115) of chicken cystatin is extended by one leucine residue (L110) as compared to the corresponding sequence in KD2 (see Figure 1); L110 forms a β-bulge of the terminal β-sheet that is conserved in family 1 and 2 cystatins (Bode et al, 1988;Stubbs et al, 1990). This difference could be responsible for non-optimal binding of the chicken cystatin-KD2 hybrids to calpain resulting in temporary inhibition.…”

Structure, alternative splicing and chromosomal localization of the cystatin-related epididymal spermatogenic gene

“…For example, the family 2 cystatin genes are characterized by their three-exon structure with conserved exon\intron splice sites and a clustering of some of these genes at the same genetic loci [2,3]. Mutation analysis [4] and X-ray crystallography [5,6] have revealed three conserved regions in the cystatin superfamily proteins that are considered to be important for the inhibition of cysteine proteases, including a glycine residue at the N-terminus, an active site sequence with conserved glutamine, valine and glycine residues, and a proline-tryptophan sequence.…”