Structure of the Drosophila apoptosome at 6.9 å resolution

Abstract

The Drosophila Apaf-1 related killer forms an apoptosome in the intrinsic cell death pathway. In this study we show that Dark forms a single ring when initiator procaspases are bound. This Dark-Dronc complex cleaves DrICE efficiently; hence, a single ring represents the Drosophila apoptosome. We then determined the 3D structure of a double ring at ∼6.9 Å resolution and created a model of the apoptosome. Subunit interactions in the Dark complex are similar to those in Apaf-1 and CED-4 apoptosomes, but there are significant differences. In particular, Dark has "lost" a loop in the nucleotide-binding pocket, which opens a path for possible dATP exchange in the apoptosome. In addition, caspase recruitment domains (CARDs) form a crown on the central hub of the Dark apoptosome. This CARD geometry suggests that conformational changes will be required to form active Dark-Dronc complexes. When taken together, these data provide insights into apoptosome structure, function, and evolution.

Figure 1. Stability and activity of Dark rings

A. Glycerol gradient profile of Dark complexes assembled and run in PSB. The peak occurs in fractions 6–8, similar to the migration of the Apaf-1 apoptosome (not shown). Also note that Dark aggregates after heating in SDS gel loading buffer to give a triplet instead of a single band. The positions of molecular weight markers (in kDa) are shown on the left. B. Proteolysis of DrICE in the absence and presence of Dark complexes. Lane 1: Dronc + DrICE in PSB; Lane 2: co-assembled Dark-Dronc complex + DrICE in PSB; p20 and p10 are cleavage products of DrICE. C. Frozen-hydrated Dark double-rings at 3 mg/ml in PSB imaged over a hole in the carbon support film. D. Dark double-rings assembled at 0.5 mg/ml in PSB and imaged on a thin carbon film E. Dark-Dronc complexes were assembled in PSB and imaged over holes at 3 mg/ml. The images show mostly single-ring aggregates as evidenced by the lack of typical side views for the double-ring and the presence of single-ring edge views.

Figure 2. Surface views of Dark double- and single-rings

A. An oblique view is shown of the Dark double-ring, which has been surface rendered in blue. This and subsequent molecular figures were made with Chimera (Goddard et al., 2005). B. The Dark double-ring is viewed edge-on along the 2-fold axis. The CARD-CARD connections are visible and a scale bar is provided. C. A top view is shown of a Dark single-ring extracted from the 3D map. In this view, the central hub within the dashed circle is dominated by ~8–10Å diameter rods. D. A bottom view of the Dark single-ring reveals the arm which connects to the β-propellers and a bridge between propellers in adjacent subunits. See also Figures S1 and S2.

Figure 3. Homology model of the Dark apoptosome

A. A linear diagram of Dark is shown with color-coded domains. B. A top view is shown of the final Dark model docked within a semi-transparent single-ring. Important features are labeled (see text for details). C. A bottom view of the final Dark model shows the excellent fit within the density map. See also Figures S3 and S4.

Figure 4. Central hub of the Dark apoptosome

A. The CARD crown is shown in a top view of the central hub, with the final model docked within the density map. An asterisk marks un-modeled density that may represent a bound small molecule. B. A top view is shown of the central hub with the CARDs omitted for clarity. NBD, HD1 and WHD from a single monomer are encircled with a dashed line and the ISM ring is indicated. C. A bottom view is shown of the central hub without the CARDs. D. A bottom view of the central hub is shown without the 3D map to clearly resolve the HD1-WHD ring. E. (left) Each CARD is linked to helix 8 and interacts with the lateral surface of its respective NBD. The model is shown within the density map. (right) Molecular interactions between a CARD (helices α1 and α4) and the NBD are shown. F. Homotypic interactions between CARDs in opposing rings mediate double-ring formation. This view is along a 2-fold axis. See also Figures S5, S6 and S7.

Figure 5. Molecular interactions in the arm and regulatory region

A. A bottom view is shown of the HD2 arm and regulatory region. The final model is docked within the semi-transparent 3D map and relevant features are labeled. B. (left) A side view is shown of the entire Dark subunit with a clear view of the HD2 arm and β-propellers, docked within the density map (right) The final model is shown in the same orientation. A molecular contact between the β7 propeller and HD1 is indicated with a black dot and the novel α28–29 loop is marked with an asterisk. C. (left) A tilted view of the Dark subunit is shown, after rotation about the horizontal axis by ~80°. (right) Features within the monomer are labeled including the α25–26 loop which forms part of the HD2 arm. The bridge density has not been modeled, though its appearance is suggestive of an extended β-hairpin that connects β8 and β7 propellers in adjacent subunits.

Figure 6. Models of Dark, Apaf-1 and CED-4 apoptosomes

A. The platform region of the octagonal Dark apoptosome is shown as a molecular ribbon diagram, viewed along the 8-fold axis in a top view. B. The Dark apoptosome is shown in a top view with the CARDs, to highlight the formation of a CARD crown on the central hub. C. The heptameric Apaf-1 apoptosome is shown in a top view with bound cytochrome c (although the position of the latter is still being refined; Yuan et al., 2010). D. (top left) A top view is shown of the octameric CED-4 apoptosome, with CARDs omitted to provide an unobstructed view of the central hub. (bottom right) The CED-4 apoptosome is shown with the double-layered CARD disk, which has 4-fold symmetry (Qi et al., 2010). See also Figure S8.

Figure 7. A comparison of lateral dimers and nucleotide binding sites in Dark, Apaf-1 and CED-4 apoptosomes

A. A lateral dimer is shown from the Dark apoptosome. Structures in panels A–C have been aligned on the leftmost NBD. B. A lateral dimer from the Apaf-1 apoptosome is shown. Differences in the packing of helix α8 are apparent. C. The CED-4 lateral dimer is shown with its truncated HD2. The conformation of the α8 helix varies in the two subunits due to differences in packing of their respective CARDs in the disk (Qi et al., 2010). D. A close-up is shown of the nucleotide binding region of Dark within the context of a lateral dimer. This view is from the bottom of the Dark single-ring. The subunit on the left has been rendered as a molecular surface to show the exposed nature of dATP. In panels D–F the lateral dimers are aligned on the rightmost NBD in each pair. The dATP binding pocket, as seen on the right, has a much shorter HD1-WHD loop than in the other apoptosomes (\marked with an asterisk). E. A close-up of the same region in an Apaf-1 lateral dimer shows a more obscured path to bound ATP on the left, and the presence of a modeled HD1-WHD loop on the right, which embraces the nucleotide from the inward surface. F. A similar close-up is shown for the atomic structure of the CED-4 dimer within the apoptosome. In this case, bound ATP is almost totally occluded within its binding pocket. See also Figure S9.

Figure 8. The presentation of CARDs on Dark and CED-4 apoptosomes

A. A tilted view is shown of the central Dark hub. The 8 CARDs in the crown are shown within calculated surfaces to provide better depth perception. B. A tilted view is shown of the CED-4 apoptosome with CARDs from the A- and B-subunits displayed within calculated surfaces. The tetrameric A-layer is shown in gold and B-layer is in silver. C. A strong clash exists between the bound CARD of the initiator procaspase (in purple) and the β-7 propeller of Dark. A monomer is shown and domains are displayed within calculated surfaces. D. This panel shows the relative orientations of A- and B-CARDs in two layers on the CED-4 apoptosome. Only CARDs and α8 helices are shown for clarity. The modeled binding of an initiator CARD (in purple) by a CED-4 CARD in the B-layer would preclude the binding of other initiator CARDs and shows significant clashes.