Atomic structure of the apoptosome: mechanism of cytochrome <i>c</i>- and dATP-mediated activation of Apaf-1

Zhou, Ming; Li, Yini; Hu, Qi; Bai, Xiao Chen; Huang, Weixin; Yan, Chuangye; Scheres, Sjors H.W.; Shi, Yigong

doi:10.1101/gad.272278.115

Cited by 209 publications

(272 citation statements)

References 43 publications

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“…S3). The central hub of the apoptosome holoenzyme displays an identical structure as that reported previously (16).…”

Section: Significancesupporting

confidence: 73%

“…Human Apaf-1 and WT caspase-9 were purified as described (16). The holoenzyme was assembled in the presence of 1 mM dATP by incubating Apaf-1, equine CytC, and caspase-9 at a molar ratio of 1:2:2.…”

Section: Methodsmentioning

confidence: 99%

“…Although this information might be crucial for understanding caspase-9 activation, neither the identities of the CARD domains nor their interactions with the apoptosome were identified. The cryo-EM structure of the apoptosome at a nearatomic resolution reveals how the monomeric Apaf-1 assembles into a functional heptameric apoptosome, but the CARD domains exhibit no obvious EM density (16).…”

mentioning

confidence: 99%

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

Zhou

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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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

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“…S3). The central hub of the apoptosome holoenzyme displays an identical structure as that reported previously (16).…”

Section: Significancesupporting

confidence: 73%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

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

Zhou

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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“…The focused classification and refinement approaches (7,8) have been used for two primary purposes: sorting out localized structural heterogeneity among particles (9,10) and improving feature resolvability of flexible domains through local refinement (11,12). These molecular machines frequently are composed of multiple protein subunits that can generate correlated and/or uncorrelated motions (13), and no experimental technique has yet captured the atomic structures of individual subunits within a single molecular machine in solution.…”

mentioning

confidence: 99%

Subunit conformational variation within individual GroEL oligomers resolved by Cryo-EM

Roh

Hryc

Jeong

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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Single-particle electron cryo-microscopy (cryo-EM) is an emerging tool for resolving structures of conformationally heterogeneous particles; however, each structure is derived from an average of many particles with presumed identical conformations. We used a 3.5-Å cryo-EM reconstruction with imposed D7 symmetry to further analyze structural heterogeneity among chemically identical subunits in each GroEL oligomer. Focused classification of the 14 subunits in each oligomer revealed three dominant classes of subunit conformations. Each class resembled a distinct GroEL crystal structure in the Protein Data Bank. The conformational differences stem from the orientations of the apical domain. We mapped each conformation class to its subunit locations within each GroEL oligomer in our dataset. The spatial distributions of each conformation class differed among oligomers, and most oligomers contained 10-12 subunits of the three dominant conformation classes. Adjacent subunits were found to more likely assume the same conformation class, suggesting correlation among subunits in the oligomer. This study demonstrates the utility of cryo-EM in revealing structure dynamics within a single protein oligomer.nderstanding the function of a molecular machine typically requires determination of the structural components at atomic or near-atomic resolution. Although X-ray crystallography can provide atomic details, the crystal lattice forces may unnaturally constrain the structure. Moreover, solution methods, such as NMR spectroscopy, can reveal short-range dynamic behavior in the absence of information on the entire complex (1, 2). Electron cryo-microscopy (cryo-EM) has made substantial progress toward achieving atomic resolution for a broad range of molecular machines by averaging thousands to millions of single-particle images that are assumed to have identical conformations (3-6).Cryo-EM has also been used to determine multiple structures of a single specimen preparation containing conformational and compositional heterogeneity (7,8). The focused classification and refinement approaches (7, 8) have been used for two primary purposes: sorting out localized structural heterogeneity among particles (9, 10) and improving feature resolvability of flexible domains through local refinement (11, 12). These molecular machines frequently are composed of multiple protein subunits that can generate correlated and/or uncorrelated motions (13), and no experimental technique has yet captured the atomic structures of individual subunits within a single molecular machine in solution. GroEL, Escherichia coli chaperonin (13,14), are composed of 14 chemically identical protomers (13,14) and have been structurally characterized by both X-ray crystallography (15, 16) and cryo-EM (5, 17-19). However, these studies have not revealed any heterogeneity among the protomers in each oligomer. Here we used a 3D classification strategy for cryo-EM data (10) to investigate structural heterogeneity among chemically identical subunits and their conformational v...

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“…The mammalian heptameric Apaf-1 apoptosome is assembled upon cytochrome c release from mitochondria. Cytochrome c binds between two β-propeller regions, destabilizing the autoinhibited monomeric form of Apaf-1, leading to the exposure and displacement of ADP for ATP or dATP and oligomerization [35,36] (Fig. 4A).…”

Section: Initial Recognition Of Pamps Is Mediated By Naipsmentioning

confidence: 99%

The NLRC4 inflammasome: The pieces of the puzzle are falling into place

Hafner-Bratkovič¹

2017

Inflammasome

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Inflammasomes are intracellular multiprotein platforms for the activation of inflammatory caspases. As components of the innate immune system, they play an important role in the fight against microbes. However, aberrant inflammasome activation has been implicated in auto-inflammatory syndromes. This review focuses on the NLRC4 inflammasome. This is perhaps not the most extensively studied, yet its mechanism of activation is by far the best understood. The NLRC4 inflammasome is activated by several proteins originating from intracellular bacteria, which are first sensed by receptors of the NAIP family. Activated NAIP binds NLRC4, which further recruits dormant NLRC4 molecules in a prion-like oligomerization event. NLRC4 enables a strong amplification of the signal, providing a fast and robust host response. The review also discusses peculiar NLRC4 inflammasome functions in promoting eicosanoid biosynthesis, actin reorganization, and its roles in autoinflammatory syndromes and sterile inflammation. Finally, the first inflammasomeindependent engagement of NLRC4 in suppressing melanoma tumor growth is presented. The emerging roles of NLRC4 in various normal and pathological processes demonstrate that there is still plenty to be learned about the NLRC4 mechanism of activation and downstream functions.

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Atomic structure of the apoptosome: mechanism of cytochrome c- and dATP-mediated activation of Apaf-1

Cited by 209 publications

References 43 publications

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

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

Subunit conformational variation within individual GroEL oligomers resolved by Cryo-EM

The NLRC4 inflammasome: The pieces of the puzzle are falling into place

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