Regulation of Caspase-3

Exosite and Allosteric Site

Caspases have similar structure of active site. Exosite that could be utilized to improve activity has been found in caspase-7 (Boucher, Blais et al. 2012). Caspase-7 also has an inhibitory allosteric site that could bind with small molecule FICA, presenting a zymogen-like conformation (Hardy, Lam et al. 2004).

Although there is no evident exosite found in caspase-3, some allosteric sites, (most of which are located on the dimer interface,) has been studied by mutagenesis. Some of mutant residues can modulate the activity of caspase-3 or even procaspase-3. The procaspase-3 was detected only little activity because the orientation of ILA (prematured L2 loop) and ILB loop cannot form an active site pocket (Bose, Pop et al. 2003).

V266E is a mutation that improves caspase-3 activity dramatically. Even in the uncleavable procaspase-3 (D5A, D26A, D175A), V266E mutant zymogen is also pseudo-activated (60-fold activity). Interestingly, V266E does not change a lot conformation around active site in the active caspase-3. Based on the crystal structure, L2’ loop is partially disorder at 185’-180’. This active procaspase-3 cannot be inhibited by endogenous XIAP like normal cleaved caspase-3. So it provides us an option for apoptosis stimuli with intrinsic efficiency.

It was found recently that many other mutant residues on the dimer interface might play an important role on inhibition of caspase-3 through manipulating the hydrogen bond or remote talking across whole dimer, like V266H, Y197C, E124A.

Post translational Modification

Natural Inhibitors

X-linked inhibitor of apoptosis proteins (XIAP) contains the second baculovirus IAP repeat domain (BIR2) targeting caspase-3 and caspase-7.

Importance of Loop Orientation

Caspases are extremely dependent on the orientation and geometry of their active site loops. If the loops are not ordered properly the enzyme fails to function. Caspase-3 has four active site loops on each half of the dimer constituting the active site bundle. Proteolytic activity is dependent on cleavage of an intersubunit linker, which releases loop 2 (L2) and L2’. . This allows L2 to make critical contacts with L3 and L4, allowing them to organize the active site, bind substrate, and orient the nucleophilic cysteine 163 (bright green) so that it can cleave after aspartate residues.

Taking a closer look at L2 and L2’ we can see a critical interaction involving aspartate 169 on L2. This residue makes two hydrogen bonds with backbone amides of V189’ and E190’, stabilizing L2 in the proper position. This reinforcement allows L2 to contact L3 so as to twist the active site cysteine into the proper orientation to attack the substrate. In addition, L2 can now contact L4 at K260. This secures L4 and allows it to make contacts in the P4 position, which greatly influence substrate specificity.

Reference

Bose, K., C. Pop, et al. (2003). "An uncleavable procaspase-3 mutant has a lower catalytic efficiency but an active site similar to that of mature caspase-3." Biochemistry 42(42): 12298-12310.

Boucher, D., V. Blais, et al. (2012). "Caspase-7 uses an exosite to promote poly(ADP ribose) polymerase 1 proteolysis." Proc Natl Acad Sci U S A 109(15): 5669-5674.

Hardy, J. A., J. Lam, et al. (2004). "Discovery of an allosteric site in the caspases." Proc Natl Acad Sci U S A 101(34): 12461-12466.