Molecular determinants of caspase-9 activation by the Apaf-1 apoptosome

Abstract

Significance Upstream cell death stimuli culminate in the activation of an initiator caspase, marking the onset of apoptosis. Activation of the initiator caspase, caspase-9, is mediated by the heptameric Apaf-1 apoptosome. How Apaf-1 apoptosome facilitates the autocatalytic activation of caspase-9 has remained controversial and largely enigmatic. Two contrasting but not mutually exclusive hypotheses, proximity-induced dimerization vs. induced conformation, emphasize different aspects of initiator caspase activation. This study provides compelling evidence to support the induced conformation model for caspase-9 activation. A previously unknown interface between Apaf-1 and caspase-9 was identified to play an essential role in caspase-9 activation, and formation of a multimeric complex between Apaf-1 caspase recruitment domain (CARD) and caspase-9 was shown to be indispensable for caspase-9 activation.

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

“…At 6.9 Å resolution, the sickle-shaped density at the edge of the central hub allowed identification and docking of two ApCARDs and one C9CARD, which are in perfect registry with the reported heterotrimeric complex (17) (Fig. 1D).…”

“…The assignment of the central CARD complex was based on the observation that two ApCARDs and one C9CARD form a stable heterotrimeric complex through both type I and type II interfaces (17) (Fig. 1B).…”

“…The fourth C9CARD associates with ApCARD through a type II interface only. Because both types of interface are known to be stable (17), assembly of the central CARD complex in this fashion maximizes the number of type I and type II interfaces, and hence is thermodynamically most favorable.…”

Caspase-9 swings both ways in the apoptosome

“…[4][5][6][7] Importantly, unlike other initiator caspases that are released from their adaptor proteins as active proteases, caspase-9 must remain bound to the apoptosome to exhibit significant catalytic activity. Furthermore, cleavage of its intersubunit linker is not required for its activation, 8 and in fact, processing of procaspase-9 to its 2-chain (p35/p12) form reduces its affinity for the apoptosome, resulting in its dissociation from the complex.…”

“…2,3 Other studies, however, suggest that the apoptosome is a holoenzyme in which oligomerized Apaf-1 acts as a positive allosteric regulator of caspase-9, promoting unique conformational changes in the protease that lead to its activation. [4][5][6][7] Importantly, unlike other initiator caspases that are released from their adaptor proteins as active proteases, caspase-9 must remain bound to the apoptosome to exhibit significant catalytic activity. Furthermore, cleavage of its intersubunit linker is not required for its activation, 8 and in fact, processing of procaspase-9 to its 2-chain (p35/p12) form reduces its affinity for the apoptosome, resulting in its dissociation from the complex.…”