Extended Substrate Recognition in Caspase-3 Revealed by High Resolution X-ray Structure Analysis Rajkumar Ganesan, Peer R. E. Mittl Stjepan Jelakovic and Markus G. Grütter⁎ Biochemisches Institut, Winterthurer Strasse 190, CH-8057, Universität Zürich, Switzerland Caspases are cysteine proteases involved in the signalling cascades of programmed cell death in which caspase-3 plays a central role, since it propagates death signals from intrinsic and extrinsic stimuli to downstream targets. The atomic resolution (1.06 Å) crystal structure of the caspase-3 DEVD-cmk complex reveals the structural basis for substrate selectivity in the S4 pocket. A low-barrier hydrogen bond is observed between the side- chains of the P4 inhibitor aspartic acid and Asp179 of the N-terminal tail of the symmetry related p12 subunit. Site-directed mutagenesis of Asp179 confirmed the significance of this residue in substrate recognition. In the 1.06 Å crystal structure, a radiation damage induced rearrangement of the inhibitor methylketone moiety was observed. The carbon atom that in a substrate would represent the scissile peptide bond carbonyl carbon clearly shows a tetrahedral coordination and resembles the postulated tetrahedral intermediate of the acylation reaction. © 2006 Elsevier Ltd. All rights reserved. *Corresponding author Keywords: caspase; substrate specificity; safety catch; low-barrier hydrogen bond; radiation damage Introduction Programmed cell death (apoptosis) is a central process in all multi-cellular organisms. This process is highly regulated and involves several different classes of proteins. Apoptosis is triggered by external and internal stimuli, which are propagated by either extrinsic or intrinsic cell death pathways. Cysteine Asp-specific proteases (caspases) are com- mon to both pathways. 1–3 In the pre-apoptotic cell caspases are synthesized as inactive pro-enzymes. Upon triggering of the cell death pathway caspases are activated by proteolytic cleavage on the C- terminal side of well-conserved aspartic acids, cleaving the polypeptide chain into p12 and p17 subunits, 4 which are in intimate contact and form one catalytic domain. Active caspases are dimers of two catalytic domains. So far 11 human caspases are known, they are classified into initiator and execu- tioner caspases, depending on their role in the cell death process. 5 Caspase-3 is a key executioner caspase. It occupies a prominent position in the apoptotic cascade where death signals from both the intrinsic and extrinsic stimuli converge to activate downstream death events. Since apoptosis is a dangerous process for the cell, caspases are highly regulated and selective towards substrates. All caspases recognize tetrapeptide sequences and have an absolute requirement for an aspartic acid in the P1 position. Caspases are subdivided into three specificity families, 6 depend- ing on their preference for P4 amino acid. The prototype caspases are caspase-1, -3 and -8 requiring tryptophan, aspartatic acid and leucine residues in the P4 positions, respectively. The specificity of caspase-3 for a P4 aspartic acid is at least 100-fold higher than for a glutamate or an asparagine. 7 Thus, the P4 selectivity is nearly as stringent as the P1 selectivity. The P4 selectivity was attributed to different interactions with residues in loops flank- ing the P4 binding pocket. 8,9 Several crystal structures of caspase-3 in complex with substrate analogues are known, which do not sufficiently explain the reason for the high selectivity in the P4 Abbreviations used: Ac, acetyl; AMC, 7-amino-4- methyl coumarin; BIR, baculovirus IAP repeat; cmk, chloromethyl ketone; LBHB, low-barrier hydrogen bond; p12, small subunit of caspase-3; p17, large subunit of caspase-3; SMAC, second mitochondrial activator of caspases; XIAP, X-linked inhibitor of apoptosis; Z/Cbz, benzyloxycarbonyl. E-mail address of the corresponding author: gruetter@bioc.unizh.ch doi:10.1016/j.jmb.2006.04.051 J. Mol. Biol. (2006) 359, 1378–1388 0022-2836/$ - see front matter © 2006 Elsevier Ltd. All rights reserved.