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. 2014 Nov 18;111(46):16254-61.
doi: 10.1073/pnas.1418000111. Epub 2014 Oct 13.

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

Qi Hu  1 , Di Wu  1 , Wen Chen  1 , Zhen Yan  1 , Chuangye Yan  1 , Tianxi He  2 , Qionglin Liang  2 , Yigong Shi  3
Affiliations

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

Qi Hu et al. Proc Natl Acad Sci U S A. .

Abstract

Autocatalytic activation of an initiator caspase triggers the onset of apoptosis. In dying cells, caspase-9 activation is mediated by a multimeric adaptor complex known as the Apaf-1 apoptosome. The molecular mechanism by which caspase-9 is activated by the Apaf-1 apoptosome remains largely unknown. Here we demonstrate that the previously reported 1:1 interaction between Apaf-1 caspase recruitment domain (CARD) and caspase-9 CARD is insufficient for the activation of caspase-9. Rather, formation of a multimeric CARD:CARD assembly between Apaf-1 and caspase-9, which requires three types of distinct interfaces, underlies caspase-9 activation. Importantly, an additional surface area on the multimeric CARD assembly is essential for caspase-9 activation. Together, these findings reveal mechanistic insights into the activation of caspase-9 by the Apaf-1 apoptosome and support the induced conformation model for initiator caspase activation by adaptor complexes.

Keywords: CARD; apoptosis; caspase activation; induced proximity; mechanism.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Oligomerized Apaf-1 CARD (i.e., ApCARD) potently activates caspase-9 to a similar level as the Apaf-1 apoptosome. (A) Caspase-9 and the heptameric ApCARD–GroES complex form a multimeric assembly. Peak fractions from the color-coded chromatograms of gel filtration (Left) were visualized on SDS/PAGE gels by Coomassie blue staining (Right). The PAGE gels are identified by the same colors as those for the gel filtration chromatograms. Blue, caspase-9 alone; green, heptameric ApCARD–GroES complex alone; red, caspase-9 plus the ApCARD–GroES complex. (B) The heptameric ApCARD–GroES or ApCARD–ClpP complex stimulates the protease activity of caspase-9 to a similar level as the preassembled Apaf1-591 apoptosome. The Apaf1-591 apoptosome has the same ability to activate caspase-9 as the full-length Apaf-1 apoptosome (26). Shown here is a representative SDS/PAGE gel. The single-chain caspase-3 (C163A) was used as the substrate. (C) The heptameric ApCARD–GroES or ApCARD–ClpP complex potently activates the caspase-9 to a similar level as the preassembled Apaf1-591 apoptosome. The fluorogenic peptide Ac-LEHD-AFC was used as the substrate. Shown here are results of the caspase-9 protease activities from the original experiments (Left) and their quantification (Right). Each experiment described in this study was independently repeated at least three times. The error bar represents the SD of the observed values.
Fig. 2.
Fig. 2.
ApCARD and C9CARD form a multimeric complex through three types of interfaces. (A) ApCARD and C9CARD form a multimeric complex on gel filtration. The apparent molecular mass of the ApCARD–C9CARD complex exceeds 75 kDa, likely containing seven or eight copies of the CARD modules. The chromatograms (Upper) and the PAGE gels (Lower) are color-coded. (B) Structure of a multimeric complex between ApCARD and C9CARD. There are six molecules of CARD in each asymmetric unit, constituting two basic repeats. Each of the two repeats contains two molecules of ApCARD and one molecule of C9CARD. Two perpendicular views are shown here. C9CARD is colored magenta, whereas ApCARD is displayed in cyan and green. (C) Each basic unit of the ApCARD–C9CARD complex uses three types of interface. The type I and type II interfaces occur between ApCARD and C9CARD, whereas the type III interface involves two ApCARD modules. The interhelical loop L12 refers to the intervening sequences between α-helices H1 and H2. Loops L23, L45, and L56 are similarly defined. (D) The type I interface was previously observed in the 1:1 complex between ApCARD and C9CARD (23). All structural figures were prepared with PyMOL (www.pymol.org).
Fig. 3.
Fig. 3.
The type II interface between ApCARD and C9CARD plays an essential role in caspase-9 activation by the Apaf-1 apoptosome. (A) Characterization of six caspase-9 variants each containing one or two missense mutations at the type II interface. The six caspase-9 variants examined here are classified into three categories in terms of phenotype: no effect (H38A and S31A/E33A), mild effect (R6A/R7A and E41A/D42A), and crippling effect (R36A and R65A). Two mutations in caspase-9 R36A and R65A, which target key residues at the type II interface, nearly abrogated the ability for these caspase-9 variants to be activated by the Apaf1-591 apoptosome. The Apaf1-591 apoptosome has the same ability to activate caspase-9 as the full-length Apaf-1 apoptosome (26). (B) Three close-up views of the interdomain H-bonds at the type II interface. Arg36 and Arg65 appear to anchor the interface by each mediating more than one H-bond. H-bonds are represented by red dashed lines.
Fig. 4.
Fig. 4.
Functional characterization of the interfaces between ApCARD and C9CARD. (A) Characterization of four Apaf1-591 variants each containing one or two missense mutations. Of the four variants, K81G/D82R targets the type II interface whereas E41K and R52G affect the type III interface. The Apaf1-591 variant K58E/K62E contains two mutations that affect residues not located at the three types of interfaces. (B) Two close-up views of the interdomain H-bonds at the type II interface (Lower Left) and type III interface (Lower Right). Unlike the other two types of interfaces, the type II interface involves two ApCARD modules.
Fig. 5.
Fig. 5.
Determination of the molar ratio between Apaf1-591 and caspase-9. (A) Determination of the molar ratio between Apaf1-591 and caspase-9 by capillary electrophoresis. A stable complex between the single-chain caspase-9 (C287A) and the Apaf1-591 apoptosome was isolated from gel filtration (Top) and visualized on SDS/PAGE by Coomassie blue staining (Middle). The peak fraction that corresponds to the caspase-9–Apaf1-591 apoptosome holoenzyme was subjected to capillary electrophoresis under denaturing condition. The peak areas were deconvoluted, integrated, and converted to molar ratio between Apaf1-591 and caspase-9. The molar ratio between Apaf1-591 and caspase-9 was determined to be 1.66 ± 0.11 or 7:4.2 ± 0.3. (B) Enzymatic characterization of caspase-9 supports the molar ratio of 7:4 between Apaf1-591 and caspase-9. Upon incubation with increasing concentrations of preassembled Apaf1-591 apoptosome, the protease activities of caspase-9 were measured and plotted against the molar ratios between Apaf1-591 and caspase-9. The highest protease activity was recorded at a molar ratio of 2:1 between Apaf1-591 and caspase-9.
Fig. 6.
Fig. 6.
A hypothetical model on the assembly of the multimeric complex between ApCARD and C9CARD. (A) An overall view of the 11-mer complex between ApCARD and C9CARD. This complex contains seven molecules of ApCARD (cyan and green) and four molecules of C9CARD (magenta). The three types of specific interfaces observed in our crystal structure (Fig. 2C) were used to generate this model. Notably, all carboxyl termini (blue spheres) of the 11 CARD domains are located on the outside surface of the helical assembly. (B) A schematic planar view of the 11-mer complex between ApCARD and C9CARD. The helical assembly is unwounded to show the interfaces among the 11 CARD molecules. There are a total of 23 interfaces, of which 14 belong to the normal three types of interface (black lines) and the other nine exhibit reversed polarity (blue lines). (C) Lys58 and Lys62 (red spheres) from four ApCARD molecules are located on the outside surface of the modeled ApCARD–C9CARD assembly. These residues appear to define a binding epitope, perhaps for caspase-9.
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References

    1. Thornberry NA, Lazebnik Y. Caspases: Enemies within. Science. 1998;281(5381):1312–1316. - PubMed
    1. Horvitz HR. Worms, life, and death (Nobel lecture) ChemBioChem. 2003;4(8):697–711. - PubMed
    1. Danial NN, Korsmeyer SJ. Cell death: Critical control points. Cell. 2004;116(2):205–219. - PubMed
    1. Yan N, Shi Y. Mechanisms of apoptosis through structural biology. Annu Rev Cell Dev Biol. 2005;21:35–56. - PubMed
    1. Riedl SJ, Shi Y. Molecular mechanisms of caspase regulation during apoptosis. Nat Rev Mol Cell Biol. 2004;5(11):897–907. - PubMed

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