Mechanistic insights into caspase-9 activation by the structure of the apoptosome holoenzyme

Abstract

Mammalian intrinsic apoptosis requires activation of the initiator caspase-9, which then cleaves and activates the effector caspases to execute cell killing. The heptameric Apaf-1 apoptosome is indispensable for caspase-9 activation by together forming a holoenzyme. The molecular mechanism of caspase-9 activation remains largely enigmatic. Here, we report the cryoelectron microscopy (cryo-EM) structure of an apoptotic holoenzyme and structureguided biochemical analyses. The caspase recruitment domains (CARDs) of Apaf-1 and caspase-9 assemble in two different ways: a 4:4 complex docks onto the central hub of the apoptosome, and a 2:1 complex binds the periphery of the central hub. The interface between the CARD complex and the central hub is required for caspase-9 activation within the holoenzyme. Unexpectedly, the CARD of free caspase-9 strongly inhibits its proteolytic activity. These structural and biochemical findings demonstrate that the apoptosome activates caspase-9 at least in part through sequestration of the inhibitory CARD domain.apoptosome | caspase-9 | holoenzyme | caspase activation | cryo-EM

Caspase-9 CARD : core domain interactions require a properly formed active site

“…We also estimated the k cat /K M of ΔCARD-His 6 against procaspase-3, and observed a similar decrease in catalytic efficiency than that of WT (Figures 2E–2G). While other studies have reported ΔCARD being more active than full-length cleaved caspase-9 (11–13), we consistently and reproducibly observed the opposite. Both the ΔCARD caspase-9 which was allowed to autoprocess at the intersubunit linker (ΔCARD-His 6 in Figure 2A) and ΔCARD caspase-9 refolded from separately expressed large and small subunits (ΔCARD in Figure 2A) were less active than full-length cleaved caspase-9.…”

“…Prior to our work there has been no data to suggest that there are existing interactions between the CARD and the catalytic core of caspase-9. However, there have been reports that point to an inhibitory role of CARD in caspase-9 activity prior to apoptosome binding, either by potentially interfering with caspase-9 homodimerization (11), or by binding to the active site and/or other regions of caspase-9 (13) to keep the enzyme in its latent state, and that Apaf-1 CARD binding relieves this inhibition. Here we observed that the CARD directly participates in stabilizing interactions with the core of caspase-9.…”

“…Although the prodomain is rarely removed intracellularly, caspase-9 was observed to be 20% more active when caspase-9’s catalytic core remains covalently linked to the CARD, as in full-length caspase-9, than when it is proteolytically removed to form the ΔCARD version of caspase-9 (9,10) (Figure 1). Later reports have shown that ΔCARD versions of caspase-9 exhibited increased activity relative to the cleaved full-length, prompting the hypothesis that CARD plays an inhibitory role in caspase-9 activity (11–13). Despite the differences in reported free caspase-9 activity, a consistent observation is that ΔCARD caspase-9 does not exhibit significant enhancement in activity in the presence of the Apaf-1 and cytochrome c. Addition of the isolated Apaf-1 CARD further enhances caspase-9 activity by five-fold, in vitro (9).…”

Human Immunodeficiency Virus Promotes Mitochondrial Toxicity

“…Prolonged or persistent PTP opening causes mitochondrial depolarization and release of cytochrome c from the mitochondrial intermembrane space. Cytochome c is required for the activation of the initiator of apoptosis, caspase-9, by forming the apoptosomic complex with Apaf-1 (Li et al 2017). Such complex activates the effector caspase-3 and triggers a feedback loop for mitochondrial disruption through cleavage of anti-apoptotic Bcl-2 family proteins (Chen et al 2007).…”

Understanding CARD Tricks in Apoptosomes

“…Almost concurrently, the Akey (Cheng et al, 2016) and Shi (Li et al, 2017) groups reported two independently determined cryo-EM structures of the CARD disk in the context of the holo-apoptosome (Figure 1C), which were refined without imposing the seven-fold symmetry and reached sufficient resolution to visualize α helices in the CARDs.…”