Caspases are involved in apoptosis and inflammation. During apoptosis, hundreds of proteins are cleaved by the executioner caspases 3 and 7, which share the same substrate recognition preference DEVD*(G/A/S). Importantly, caspase-7 is intrinsically less active than caspase-3, at least on small peptidic substrates, but is more proficient at proteolyzing the poly(ADP ribose) polymerase 1 (PARP) because its N-terminal domain (NTD) contains a substrate exosite. Despite a lower intrinsic activity however, few substrates of executioner caspases are more efficaciously cleaved by caspase-7, including the Hsp90 cochaperone p23. The cleavage of p23 is aided by the NTD, but the precise molecular determinants governing this substrate-peptidase interaction are poorly understood.
To better comprehend the contribution of the NTD of caspase-7 in enhancing the cleavage of p23, series of deletion and substitution mutants were generated and tested for enzymatic activity. Although we demonstrate that the critical lysine residues that were shown to participate in the exosite for PARP also contribute to efficacious p23 cleavage, other determinants participate in this exosite. Of note, globally no key amino acids were clearly identified suggesting that the exosite is cryptic and likely involve a series of weak interactions distributed over several residues of the NTD. We also performed nuclear magnetic resonance titration experiments using purified p23 and a short peptide from the NTD (residues 34-47) to identify determinants of the substrate that interact with the exosite. Analysis of the 1H-15N-HSQC spectra identified several residues of p23 (D23, S44, D45, F47, H49) which magnetic environment change upon peptide addition. These residues all localize to the same region, which is structurally far away from the C-terminal caspase-7 cleavage site.
Globally, our works suggest that the NTD of caspase-7 has evolved to assist the proteolysis of many substrates using different molecular determinants. These studies are supported by FRQ-NT and NSERC.