THE THREE-DIMENSIONAL STRUCTURE OF APOPAIN/CPP32, A KEY MEDIATOR OF APOPTOSIS. Jennifer Rotonda and Joseph W. Becker, Department of Biochemistry, Merck Research Laboratories, PO Box 2000, Rahway, New Jersey 07065
Cysteine proteases related to mammalian interleukin-1[[beta]] converting enzyme (ICE) and to its C. elegans homologue, CED-3, play a critical role in the biochemical events that culminate in apoptosis. We have determined the three-dimensional structure of a complex of the human CED-3 homologue CPP32/apopain with a potent tetrapeptide-aldehyde inhibitor.1 The protein resembles ICE in overall structure, but its S4 subsite is strikingly different in size and chemical composition. These differences account for the variation in specificity between the ICE- and CED-3-related proteases and enable the design of specific inhibitors that can probe the physiological functions and disease states with which they are associated.
We have solved the three-dimensional structure of apopain in complex with the peptide-aldehyde inhibitor Ac-DEVD-CHO at a nominal resolution of 2.5 Å. The crystals belong to the orthorhombic space group I222 with a=69.81, b=84.62, c=96.79 Å with one enzyme:inhibitor complex per asymmetric unit. Three-dimensional diffraction data extending to a resolution of 2.5 Å were collected at room temperature using a Siemens area detector and CuK[[alpha]] radiation from a Rigaku RU-200 rotating-anode X-ray generator. 20,801 observations of 8,929 unique reflections were merged with an R-factor of 5.55%. The structure was solved by molecular replacement, using X-PLOR and a model based on the protein component of PDB entry 1ICE, the structure of ICE:Ac-YVAD-CHO complex. The current model was constructed by interactive model-building and refined using X-PLOR. In early stages of model-building, phase refinement using SQUASH significantly improved the quality of electron density maps. The R-factor of the refined model is 19.5% (Rfree=27.5%) and the stereochemistry is reasonable (r.m.s. deviation of bonds =0.007 Å, angles = 1.31[[ring]]).
1. J. Rotonda, D. W. Nicholson, K. M. Fazil, M. Gallant, Y. Gareau, M. Labelle, E. P. Peterson, D. M. Rasper, R. Ruel, J. P. Vaillancourt, N. A. Thornberry and J. W. Becker (1996) Nature Structural Biology, in press.