Caspase-8 specificity probed at subsite S-4: Crystal structure of the caspase-8-Z-DEVD-cho complex

Caspase-8 is an initiator enzyme in the Fas-mediated pathway of which the d ownstream executioner caspase-3 is a physiological target. Caspases are cys teine proteases that are specific for substrates with an aspartic acid resi due at the P-1 position and have an optimal recognition motif that incorpor ates four amino acid residues N-terminal to the cleavage site. Caspase-8 ha s been classified as a group III caspase member because it shows a preferen ce for a small hydrophobic residue at the P-4 substrate position. We report the X-ray crystallographic structure of caspase-8 in complex with benzylox yearbonyl-Asp-Glu-Val-Asp-aldehyde (Z-DEVD), a specific group II caspase in hibitor. The structure shows that the inhibitor interacts favourably with t he enzyme in subsite S-4. Kinetic data reveal that Z-DEVD (K-i 2 nM) is an almost equally potent inhibitor of caspase-8 as the specific group III inhi bitor Boc-IETD-aldehyde (K-i 1 nM). In view of this finding, the original c lassification of caspases into three specificity groups needs to be modifie d, at least for caspase-8, which tolerates small hydrophobic residues as we ll as the acidic residue Asp in subsite S-4. We propose that the subsite S- 3 must be considered as an important specificity-determining factor. (C) 20 00 Academic Press.