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. 2017 Jan 3;25(1):40-52.
doi: 10.1016/j.str.2016.11.002. Epub 2016 Dec 1.

A Near-Atomic Structure of the Dark Apoptosome Provides Insight into Assembly and Activation

Tat Cheung Cheng  1 , Ildikó V Akey  1 , Shujun Yuan  2 , Zhiheng Yu  3 , Steven J Ludtke  4 , Christopher W Akey  5
Affiliations

A Near-Atomic Structure of the Dark Apoptosome Provides Insight into Assembly and Activation

Tat Cheung Cheng et al. Structure. .

Abstract

In Drosophila, the Apaf-1-related killer (Dark) forms an apoptosome that activates procaspases. To investigate function, we have determined a near-atomic structure of Dark double rings using cryo-electron microscopy. We then built a nearly complete model of the apoptosome that includes 7- and 8-blade β-propellers. We find that the preference for dATP during Dark assembly may be governed by Ser325, which is in close proximity to the 2' carbon of the deoxyribose ring. Interestingly, β-propellers in V-shaped domains of the Dark apoptosome are more widely separated, relative to these features in the Apaf-1 apoptosome. This wider spacing may be responsible for the lack of cytochrome c binding to β-propellers in the Dark apoptosome. Our structure also highlights the roles of two loss-of-function mutations that may block Dark assembly. Finally, the improved model provides a framework to understand apical procaspase activation in the intrinsic cell death pathway.

Keywords: apoptosis; apoptosome; procaspase activation; programmed cell death; single particle cryo-EM.

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Figures

Figure 1
Figure 1. Overview of double and single Dark rings
(A) A top view of the combined 3D density map is shown with the top ring in blue, the bottom ring in yellow and a single Dark subunit in grey. V-shaped domains are from a map obtained with focused 3D classification at ~7.3 Å resolution. (B) An oblique view is shown of the composite 3D density map. (C, D) Top and bottom views of a single octagonal ring are shown. A V-shaped domain with β-propellers (β7, β8) is indicated.
Figure 2
Figure 2. Ribbon model of a single Dark ring
(A, B) A top view is shown of a single ring, without and with and the composite 3D density map. Dark domains are color-coded (see color key). The central hub is located within a dashed circle and the CARD crown is indicated. (C, D) Bottom views of the final model are shown and distinctive features are labeled.
Figure 3
Figure 3. Novel domain features in the Dark apoptosome
(A,B) The CARD-NBD-HD1 module within the central hub is shown in the absence (panel A; this work) and presence of bound Dronc CARD (panel B; PDB 39JK; Pang et al., 2015). Three predicted α-helices have been replaced with extended strands as indicated in our model (panel A). For the structure in panel B, Dark was forced to assemble with a bound nucleotide diphosphate (modeled as ADP) by the addition of a Dronc CARD. (C) Strands 17 and 19 in HD1 have replaced α-helices that are present in other family members and are shown in the density map. (D) The signature β-hairpin in the WHD is shown (Ser409-Q414) as part of a small β-sheet that is dominated by 5 prolines (Pro376, 377, 382, 415, and 423).
Figure 4
Figure 4. Interactions within the central hub
(A) A top view is shown of the central hub. The ISM ring is indicated by a dashed orange circle, while the HD1-WHD ring is enclosed within a zoned map. (B) A close-up is shown of the α-12/α-13 helical pair from the ISM ring. A hydrophobic zipper is present between the two α-helices. Side chain density in the map is shown in the left panel, while residues in the zipper are labeled in the right hand panel. (C) A ribbon model of the HD1-WHD ring is shown and one interface between adjacent subunits is indicated with a dashed oval. (D) Interactions within the boxed area on the right are highlighted. (E, F) A D333Q point mutation may knock out a three-way interaction at the WHD-HD1 subunit interface. (G) A V316I point mutation would block interactions of HD1 with an un-modeled main chain density (marked with black dots) that originates from a loop in the 7-blade β-propeller.
Figure 5
Figure 5. dATP binding in the Dark apoptosome
(A) The dATP binding pocket is located at the interface of the NBD and HD1. Bound dATP is shown in ball and stick representation. (B) The dATP binding pocket is shown within the density map. Some hydrogen bonds and salt bridges are indicated by dashed lines. Arginine 322 from HD1 may act as a sensor for dATP. middle inset: The fit of dATP in the density map is shown at a higher threshold with the adenine base in an anti configuration. (C) The binding pocket for the adenine ring is formed primarily by hydrophobic residues, whose side chains are shown in ball and stick representation. (D) Interactions of dATP are summarized in a molecular schematic. Hydrogen bonds and salt bridges are shown by dashed lines and ring stacking interactions are indicated with capped solid lines.
Figure 6
Figure 6. HD2 and β-propeller interactions
(A) Domains within the V-shaped region are shown as ribbons within the improved density map. (B) Helices in HD2 form an apex upon which the two β-propellers rest and stabilize the V-shaped domain. Individual α-helices and loops in HD2 are labeled. (C, D) Calculated molecular surfaces are shown for V-shaped domains in human (panel C) and Dark apoptosomes (panel D). Cytochrome c (in red) is tightly bound to the 8-blade β-propeller of Apaf-1. A similar tight interaction with Dark would leave a large gap relative to the 7-blade β-propeller, which has rotated outwards (see arrow). (E, F) Side views are shown of the models in panels C and D. Note that human HD2 is not shown in panels D and F.
Figure 7
Figure 7. Adenylylation of the NBD and caged micelles
(A) A novel adenylylation of Lys251 is present in Dark. Hydrogen bonds and π-cation interactions to Lys218 are indicated with dashed lines. At this threshold the Lys218-adenine ring interaction is visible. Inset bottom left: The Lys251 modification is shown in a reverse view at a higher density threshold. (B) DHPG/NP-40 mixed micelles are caged between opposing rings in the Dark apoptosome. Micelle densities were low pass filtered to ~15 Å and four of these are viewed along a two-fold axis in this cut-away view. Top: density map; Bottom: map with docked model. (C) A view along an alternate 2-fold axis reveals micelle interactions with the CARD-NBD loop (Gln98) and helix α8 (Ser100) from opposing NBDs.
Figure 8
Figure 8. CARDs in ground state and active apoptosomes
(A, B) Top views are shown of Dark and Apaf-1 apoptosomes in the ground state (left: this work, PDB 5JUL; right: Zhou et al., 2015; PDB 3JBT). A single subunit is color coded and the CARD crown and cytochrome c molecules are labeled and color-coded in green and red, respectively. ISM rings in the apoptosomes are marked with a dashed circle (red). (C) A zoomed in view is shown of a post-activation Dark apoptosome (Pang et al., 2015; PDB 3J9K). CARD-CARD heterodimer formation by Dark and Dronc creates an expanded crown that does not perturb intrinsic Dark CARD-NBD interactions. (D) A similar view is shown of the active human apoptosome (Cheng et al., 2016, PDB 5JUY). The offset of an acentric 8-CARD disk is apparent. The spiral-shaped CARD disk differs dramatically in location and organization relative to the expanded CARD crown in the post-activation Dark apoptosome shown in panel C.
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