Structural and Enzymatic Insights into Caspase-2 Protein Substrate Recognition and Catalysis

Abstract

Caspase-2, the most evolutionarily conserved member in the human caspase family, may play important roles in stress-induced apoptosis, cell cycle regulation, and tumor suppression. In biochemical assays, caspase-2 uniquely prefers a pentapeptide (such as VDVAD) rather than a tetrapeptide, as required for efficient cleavage by other caspases. We investigated the molecular basis for pentapeptide specificity using peptide analog inhibitors and substrates that vary at the P5 position. We determined the crystal structures of apo caspase-2, caspase-2 in complex with peptide inhibitors VDVAD-CHO, ADVAD-CHO, and DVAD-CHO, and a T380A mutant of caspase-2 in complex with VDVAD-CHO. Two residues, Thr-380 and Tyr-420, are identified to be critical for the P5 residue recognition; mutation of the two residues reduces the catalytic efficiency by about 4-and 40-fold, respectively. The structures also provide a series of snapshots of caspase-2 in different catalytic states, shedding light on the mechanism of capase-2 activation, substrate binding, and catalysis. By comparing the apo and inhibited caspase-2 structures, we propose that the disruption of a non-conserved salt bridge between Glu-217 and the invariant Arg-378 is important for the activation of caspase-2. These findings broaden our understanding of caspase-2 substrate specificity and catalysis.

Caspase Substrates and Inhibitors

“…Interestingly, only caspase 2 and caspase 9 cleaved their putative preferred substrates (VDVAD and LEHD respectively) the most efficiently and did not recognize other substrates. The findings are in line with reports describing that for efficient cleavage caspase 2 requires P5 residue in the substrate (Talanian et al, 1997; Tang et al, 2011). Caspase 8 and 10 preferred the substrate sequence of caspase 9 (LEHD) rather than their assumed substrate of IETD sequence, although both substrates were hydrolyzed well.…”

Mechanistic and Structural Understanding of Uncompetitive Inhibitors of Caspase-6

“…As for the inability of 3 to inhibit Lamin A cleavage, the presence of substrate residues more distal to the scissile bond (P5–P8) may alter the general conformation of the inhibitor binding site to disrupt the key L2–L4 interactions observed in Figure 5B. The importance of P5 for caspase-2 substrate recognition and catalysis has been described [31] and we speculate that the inhibitor binding site defined here may be altered by similar enzyme-substrate interactions.…”

TRIM16 overexpression induces apoptosis through activation of caspase-2 in cancer cells

“…Inactive initiator caspases with long pro-domains contain specific protein–protein interaction motifs, which suggest these proteolytic enzymes are regulated prior to cleavage and subsequent activation [28, 30]. A physical interaction of TRIM16 with procaspase-2 in the cytoplasm of MCF7 cells suggests that TRIM16 may have a central role upstream of caspase-2 activation.…”