Crystal Structure of Full-Length Apaf-1: How the Death Signal Is Relayed in the Mitochondrial Pathway of Apoptosis

Abstract

The apoptotic protease-activating factor 1 (Apaf-1) relays the death signal in the mitochondrial pathway of apoptosis. Apaf-1 oligomerizes on binding of mitochondrially released cytochrome c into the heptameric apoptosome complex to ignite the downstream cascade of caspases. Here, we present the 3.0 Å crystal structure of full-length murine Apaf-1 in the absence of cytochrome c. The structure shows how the mammalian death switch is kept in its "off" position. By comparing the off state with a recent cryo-electron microscopy derived model of Apaf-1 in its apoptosomal conformation, we depict the molecular events that transform Apaf-1 from autoinhibited monomer to a building block of the caspase-activating apoptosome. Moreover, we have solved the crystal structure of the R265S mutant of full-length murine Apaf-1 in the absence of cytochrome c to 3.55 Å resolution and we show that proper function of Apaf-1 relies on R265 in the vicinity of the bound nucleotide.

Structural Determinants at the Interface of the ARC2 and Leucine-Rich Repeat Domains Control the Activation of the Plant Immune Receptors Rx1 and Gpa2

“…7B, 3). The recently resolved crystal structure of the autoinhibited murine Apaf1 shows that the WD40 sensor domain in Apaf1 functions like a clamp by interacting with both the NB and helical domain II (ARC3, absent in NB-LRR R proteins; Reubold et al, 2011). Binding of cytochrome c changes the overall structure of the WD40 sensor and breaks its interaction with the NB, enabling or even forcing the NB and ARC1 to rotate to the open, ATP-bound conformation (Fig.…”

Role of the salt bridge between glutamate 546 and arginine 907 in preservation of autoinhibited form of Apaf-1

“…It is normally found in the cytosol in its auto-inhibited (closed) form (Zou et al, 1997). Crystal structure of full-length murine Apaf-1, in the absence of cytochrome c at 3.55 A° resolution has recently been determined (Reubold et al, 2011). It has been demonstrated earlier, that the role of WRDdomain at the c-terminal of Apaf-1 is to stabilize it in its auto-inhibited form, because mutant of Apaf-1 lacking WRD-domain, spontaneously assembles and form apoptosome, even in the absence of cytochrome c (Hu et al, 1998;Srinivasula et al, 1998).…”

“…In the absence of cytochrome c, WD1 interacts with NOD and prevents from conformational changes in NOD and CARD domains, which are essential for apoptosome formation. In the absence of WRD, NOD domain is free for conformational changes (Reubold and Eschenburg, 2012;Reubold et al, 2011). The structural data suggests that cytochrome c binding leads to rotation of WD1 upward and it breaks salt bridges between WD1 and NOD domains.…”

“…NOD in the middle of Apaf-1has four sub-domains including NBD (nucleotide-binding domain), HD1 (helical domain 1), WHD (winged-helix domain), and HD2 (helical domainn2) that serve as binding site for nucleotide and apoptosome oligomerization. WRD domain at the c-terminus of Apaf-1 has thirteen WD40 repeats and it composes two β-propeller including WD1 and WD2 (Reubold et al, 2011;Yu et al, 2005;Yuan et al, 2010). One of the main questions regarding apoptosome formation is how can Apaf-1 preserve its auto-inhibited form in the absence of cytochrome c. It has been shown mutant Apaf-1 lacking WRD can activate procaspase-9 in the absence of cytochrome c and dATP.…”

A Systems Biology Analysis of Apoptosome Formation and Apoptosis Execution Supports Allosteric Procaspase-9 Activation

“…Apaf-1 is a member of the STAND (signal transduction ATPases with numerous domains) family (9) and comprises three functional domains: a NH 2 -terminal caspase recruitment domain (CARD), a nucleotide-binding and oligomerization domain, and a COOH-terminal WD40 repeat domain (4,10). Upon activation of the mitochondrial apoptosis pathway, cytochrome c is released into the cytosol and activates Apaf-1, and active Apaf-1 then homo-oligomerizes into the heptameric apoptosome backbone (11,12). In the fully assembled complex, the CARDs of Apaf-1 form a ring structure that sits above the central hub of the apoptosome (13), and interactions with the CARD in the procaspase-9 prodomain recruit procaspase-9 into the apoptosome (14).…”