Apoptosis is a highly orchestrated form of cell death that rely on a family of protease called caspases. During this process, hundreds of proteins are cleaved by caspases, most of them by the executioner caspase-3. Despite a lower intrinsic activity, caspase-7, which shares the same substrate primary sequence preference as caspase-3, is better at cleaving some substrates like poly(ADP ribose) polymerase 1 (PARP) and Hsp90 cochaperone p23. Previous studies suggested the existence of molecular determinant located outside the substrate binding pocket (exosite) to explain this difference but failed to identify them.
Here, using various biochemical essays, we identified an exosite for PARP and p23 located in caspase-7 's N-terminal domain. We identified key lysine residues (K(38)KKK) within the N-terminal domain of caspase-7 as critical elements for the efficient proteolysis of these two substrates. Caspase-7's N-terminal domain binds PARP and improves its cleavage by a chimeric caspase-3 by ∼30-fold. Cellular expression of caspase-7 lacking the critical lysine residues resulted in less-efficient PARP and p23 cleavage compared with cells expressing the wild-type peptidase. We further showed, using a series of caspase chimeras, the positioning of p23 on the enzyme providing us with a mechanistic insight into the binding of the exosite.
In summary, we have uncovered a role for the N-terminal domain (NTD) and the N-terminal peptide of caspase-7 in promoting key substrate proteolysis. Moreover, the usage of exosite by caspases suggest a new way to target those protease more specifically.