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, 29 (22), 2349-61

Atomic Structure of the Apoptosome: Mechanism of Cytochrome C- And dATP-mediated Activation of Apaf-1

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Atomic Structure of the Apoptosome: Mechanism of Cytochrome C- And dATP-mediated Activation of Apaf-1

Mengying Zhou et al. Genes Dev.

Abstract

The apoptotic protease-activating factor 1 (Apaf-1) controls the onset of many known forms of intrinsic apoptosis in mammals. Apaf-1 exists in normal cells as an autoinhibited monomer. Upon binding to cytochrome c and dATP, Apaf-1 oligomerizes into a heptameric complex known as the apoptosome, which recruits and activates cell-killing caspases. Here we present an atomic structure of an intact mammalian apoptosome at 3.8 Å resolution, determined by single-particle, cryo-electron microscopy (cryo-EM). Structural analysis, together with structure-guided biochemical characterization, uncovered how cytochrome c releases the autoinhibition of Apaf-1 through specific interactions with the WD40 repeats. Structural comparison with autoinhibited Apaf-1 revealed how dATP binding triggers a set of conformational changes that results in the formation of the apoptosome. Together, these results constitute the molecular mechanism of cytochrome c- and dATP-mediated activation of Apaf-1.

Keywords: Apaf-1; apoptosis; apoptosome; caspase activation; caspase-9; cryo-EM structure.

Figures

Figure 1.
Figure 1.
Overall structure of the Apaf-1 apoptosome. (A) An overall view of the EM density for the Apaf-1 apoptosome. The resolution is color-coded for different regions of the apoptosome. The surface view of the apoptosome is shown here. The resolution goes to 3.0–3.5 Å in the central hub of the apoptosome. (B) Two close-up views of the EM density surrounding CytC. The two β propellers WD1 and WD2 consist of WD40 repeats 1–7 and 8–15, respectively. (C) Overall structure of the Apaf-1 apoptosome. Two views are shown. The top face refers to the CytC-exposed side of the apoptosome disk. CytC is colored yellow, and the domains within each Apaf-1 protomer are color-coded. A and B were prepared using Chimera (Pettersen et al. 2004). Except for Figures 5 and 6, all other figures were prepared using PyMol (http://www.pymol.org).
Figure 2.
Figure 2.
Structure of an activated Apaf-1 protomer in the apoptosome. (A) Structure of an activated Apaf-1 protomer in the apoptosome is shown in cartoon representation (left panel) and electrostatic surface potential (right panel). Three boxed interdomain interfaces are detailed in BD. (B) A close-up view of the interface between WHD and the NBD–HD1 module. Potential hydrogen bonds are represented by red dashed lines. (C) A close-up view of the interface between WHD and HD2. This interface contains a large number of van der Waals contacts. (D) A close-up view of the interface between HD2 and the two β propellers. The intensity of interaction between HD2 and WD2 is considerably stronger than that between HD2 and WD1. The bottom panel zooms in on the van der Waals interactions mediated by three hydrophobic residues from HD2: Leu595, Trp597, and Ile603.
Figure 3.
Figure 3.
Interactions between two adjacent Apaf-1 protomers in the apoptosome. (A) An overall view of two adjacent Apaf-1 protomers in the apoptosome. The top face is shown. The domains are color-coded. (B) A focused view of the domains that mediate intermolecular interactions. An Apaf-1 protomer uses its NBD and WHD to contact the NBD and HD1, respectively, of an adjacent Apaf-1 protomer. The interactions are detailed in C and D. (C) A close-up view of the interface between two neighboring NBDs. This interface contains a large number of H-bonds (red dashed lines). An inset is shown to highlight a network of closely spaced H bonds. (D) A close-up view of the interface between the WHD of one Apaf-1 protomer and HD1 of an adjacent Apaf-1 protomer.
Figure 4.
Figure 4.
Recognition of CytC by the WD1 and WD2 domains of Apaf-1. (A) CytC is sandwiched between WD1 and WD2 with excellent complementarity in shape and charge. CytC and WD1/WD2 are shown in electrostatic surface potential. (B) A schematic diagram of the interactions between CytC and the WD1/WD2 domains. The three boxed regions are detailed in CE. (C) A close-up view of the interface between CytC and WD2. This interface contains three putative intermolecular H bonds (red dashed lines). Notably, Gly56 contributes a H bond and makes van der Waals contacts to WD2. (D) A close-up view of the interface between CytC and the adjoining area of WD2 and WD1. Pro76 and Ile81 make van der Waals contacts to neighboring structural elements from WD2 and WD1, respectively. Lys72 donates a H bond to Asp902 of WD1. (E) A close-up view of the interface between CytC and WD1. Gln12 of CytC likely makes a H bond to Glu700 of WD1.
Figure 5.
Figure 5.
Biochemical characterization of the interactions between Apaf-1 and CytC. (A) Wild-type CytC forms a stable complex with the full-length Apaf-1. Three gel filtration chromatograms are shown in the left panel, and the corresponding fractions from gel filtration are shown on SDS-PAGE gels in the right panels. To clearly visualize the results, the baselines for the magenta (CytC alone) and green (Apaf-1 + wild-type CytC) chromatograms were shifted by 25 and 50 mAU, respectively. (B) Wild-type CytC binds to the full-length Apaf-1 with an association constant of ∼2.04 × 106 M−1 ± 0.25 × 106 M−1, which corresponds to a binding affinity of ∼0.49 µM ± 0.06 µM. Shown here are the raw data from ITC (left panel) and curve fitting (right panel). (C) Assessment of the interactions between five representative CytC mutants and Apaf-1 by gel filtration. To better display the results, the baselines for the five CytC mutants were shifted incrementally by 25 mAU each. Only wild-type CytC and the K87W mutant retained stable association with Apaf-1.
Figure 6.
Figure 6.
Impact of CytC mutations on apoptosome assembly and caspase-9 activation. (A) Impact of CytC mutations on caspase-9 activation in an in vitro cleavage assay using caspase-3 (C163A) as the substrate. For each CytC mutant, only the residue number and the target residue are labeled. For example, “56K” denotes G56K. The only double mutation (DM) denotes K79F and I81W. (B) Impact of these CytC mutations on caspase-9 activation in a second in vitro cleavage assay using the fluorogenic peptide Ac-LEHD-AFC as the substrate. (C) Assessment of the impact of representative CytC mutations on apoptosome formation by gel filtration. The wild-type control is shown in the top panel, where the baselines for the green (Apaf-1 alone) and red (CytC alone) chromatograms were shifted by 25 and 50 mAU, respectively. The five representative CytC mutants are displayed in the bottom panel, where the baselines of the chromatograms were shifted incrementally by 25 mAU each. Notably, the CytC-K72W mutant partially retained the ability to form an apoptosome.
Figure 7.
Figure 7.
Conformational differences between the autoinhibited Apaf-1 and the activated Apaf-1 protomer from the apoptosome. (A) Structural overlay of the autoinhibited Apaf-1 and the activated Apaf-1 protomer on their respective NBD–HD1 modules. (B) A close-up view of the two WHDs derived from A. These two WHDs are related to each other by a pseudo-twofold symmetry axis. (C) Superposition of the two WHDs results in near-perfect alignment of the WHD–HD2–WD2 rods from the two Apaf-1 molecules. (D) A close-up view of the WD1 and WD2 derived from C. Relative to the autoinhibited Apaf-1, WD1 in the activated Apaf-1 promoter undergoes a rotation of ∼60° toward the WD2. This movement is triggered by CytC binding. (E) Movement of WD1 would cause severe steric clashes with the NBD in the autoinhibited conformation of Apaf-1. This finding explains why, in addition to WD1, CytC binding must induce conformational changes in other domains of Apaf-1.
Figure 8.
Figure 8.
Comparison of nucleotide binding between the autoinhibited Apaf-1 and the activated Apaf-1 protomer from the apoptosome. (A) An overall comparison of the domains that contribute to nucleotide binding. (Left panel) In the activated Apaf-1 protomer, dATP is bound exclusively by NBD and HD1 and exposed to solvent. (Right panel) In the autoinhibited Apaf-1, ADP is buried and bound at the interface of NBD, HD1, and WHD. (B) A close-up comparison of nucleotide binding in these two Apaf-1 molecules. (Left panel) In the activated Apaf-1 protomer, dATP is recognized through a number of H bonds mediated by the Sensor I Arg265, the Walker B residue Asp244, the P-loop residue Lys160, and other main chain groups. No residue from WHD is involved in coordinating dATP. (Right panel) In the autoinhibited Apaf-1, ADP is recognized in part by the MHD/LHD residue His438 through a H bond.
Figure 9.
Figure 9.
Mechanism of Apaf-1 activation and apoptosome assembly. In the absence of apoptotic stimuli, Apaf-1 exists in cells as an ADP-bound, autoinhibited monomer. At the onset of apoptosis, CytC is released into the cytoplasm, where it binds to the WD2 and WD1 domains of Apaf-1 with a dissociation constant of ∼0.49 µM. CytC binding brings WD1 closer to WD2 and pushes the NBD–HD1 module away, resulting in the departure of WHD from the nucleotide-binding interface and subsequent exposure of the bound ADP to solvent. Such changes may greatly facilitate displacement of ADP by dATP or ATP, which favors the activated conformation of Apaf-1. Seven molecules of activated Apaf-1 assemble into a closed apoptosome.

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