Slow folding and assembly of a procaspase-3 interface variant.

ABSTRACT

Caspases execute apoptosis and exist in the cell as inactive zymogens (procaspases) prior to activation. Initiator procaspases are monomers that must dimerize for activation, while effector procaspases, such as procaspase-3, are stable dimers that must be processed for activation. The dimer interface regions of the two subfamilies are different, although the role of the interface in oligomerization is not known. Equilibrium and kinetic folding studies were performed on procaspase-3(C163S,V266H), an interface variant, to determine the importance of the dimer interface in the folding of procaspase-3. Equilibrium folding data at pH 5 and 7 display a hysteresis, indicating a kinetically controlled folding reaction. Refolding kinetic studies reveal a complex burst phase, followed by a series of monomeric intermediates. At longer refolding times, the monomer populates a species that becomes kinetically trapped and slowly aggregates. Unfolding kinetic studies reveal a hyperfluorescent native ensemble that unfolds to form highly structured monomeric intermediates that unfold very slowly. Dimerization is very slow, likely because of the inability to correctly orient the histidine residues in the interface, so the initial encounter complex for dimerization is inefficient. As a consequence, the monomer folds into species that aggregate. Introducing a histidine into the interface of procaspase-3 prevents activation by acting as a negative design element, providing evidence that the interface region is a site of regulation of caspase assembly in general by affecting the rate of dimerization.