Removal of the pro-domain does not affect the conformation of the procaspase-3 dimer.

We have investigated the oligomeric properties of procaspase-3 and a mutant that lacks the pro-domain (called pro-less variant). In addition, we have examined the interactions of the 28 amino acid pro-peptide when added in trans to the pro-less variant. By sedimentation equilibrium studies, we have found that procapase-3 is a stable dimer in solution at 25 degrees C and pH 7.2, and we estimate an upper limit for the equilibrium dissociation constant of approximately 50 nM. Considering the expression levels of caspase-3 in Jurkat cells, we predict that procaspase-3 exists as a dimer in vivo. The pro-less variant is also a dimer, with little apparent change in the equilibrium dissociation constant. Thus, in contrast with the long pro-domain caspases, the pro-peptide of caspase-3 does not appear to be involved in dimerization. Results from circular dichroism, fluorescence anisotropy, and FTIR studies demonstrate that the pro-domain interacts weakly with the pro-less variant. The data suggest that the pro-peptide adopts a beta-structure when in contact with the protein, but it is a random coil when free in solution. In addition, when added in trans, the pro-peptide does not inhibit the activity of the mature caspase-3 heterotetramer. On the other hand, the active caspase-3 does not efficiently hydrolyze the pro-domain at the NSVD(9) sequence as occurs when the pro-peptide is in cis to the protease domain. Based on these results, we propose a model for maturation of the procaspase-3 dimer.