A CARD-CARD disk on the pc-9 apoptosome
We recently reported the structure of a human apoptosome with bound pc-9 CARDs (Yuan et al., 2010
). To prepare this complex, we inserted a thrombin site in the linker between the CARD and p20 domain of pc-9. We then engineered single and triple mutants that removed known cleavage sites in the p20-p10 linker to create pc-9t (D315A) and pc-9tm (E306A/D315A/D330A). After assembling apoptosomes with pc-9tm, a single clip by thrombin released the pc-9 catalytic domains (p20-p10) in low salt buffer (LSB; Yuan et al., 2010
). This created a pc-9 CARD apoptosome that was less prone to aggregation than the pc-9 complex. Procaspase-9 and Apaf-1 CARDs formed a disk-like feature that sits above the heptameric platform in this complex (Yuan et al., 2010
; Figures S1A, S1B
). However, the disk-like feature may be blurred because it is linked to the platform by potentially flexible, CARD-NBD linkers.
To further characterize the CARD-CARD disk and its role in pc-9 activation, we determined the stoichiometry of pc-9 to Apaf-1 in active apoptosomes with quantitative mass spectrometry. In one experiment, apoptosomes were assembled with an excess of pc-9 (D315A) molecules that contained a TEV site within the CARD-p20 linker (pc-9TEV). Complexes were purified on a 10-40% glycerol gradient (Figure S2A
, lane 8) and a peak fraction was used to generate tryptic peptides, which were identified by tandem mass spectrometry (LC/MS-MS) (Experimental Procedures). The peptide MS/MS total ion current (TIC) signal intensities from each protein were averaged together to estimate the amount of each component in the complex (Asara et al., 2008
; Jiang et al., 2010
). With this approach, the ratio of pc-9TEV to Apaf-1 was found to be ~0.8 : 1 (Figure S2C
). As a control, the observed ratio of Apaf-1 to cytochrome c was ~1 : 1.15 (Acehan et al., 2001). In a second experiment, we used glycerol gradients to determine the amounts of pc-9tm and cytochrome c that were just sufficient to saturate binding to the apoptosome. Peak fractions of this complex are shown in Figure S2B
. We then assembled pc-9tm apoptosomes and analyzed the complexes by quantitative mass spectrometry (Figure S2C
). The measured ratio for pc-9, Apaf-1 and cytochrome c in the complexes was ~0.8 : 1 : 1.6. Based on the data, we surmise that the apoptosome may bind ~5-7 pc-9 monomers to form the CARD-CARD disk in the active complex.
We then analyzed ~1000 side views of double-ring apoptosomes, which occurred at a frequency of ~2% in images of frozen-hydrated, pc-9 CARD apoptosomes (Yuan et al., 2010
). In total, 20 side-view classes were obtained with EMAN (Ludtke et al., 1999
) and 8 representative class averages are shown in . Two striking observations can be made from the classes. First, double-ring particles are formed by interactions between CARD-CARD disks, which are sandwiched between opposing platforms. From the images, a single disk is estimated to be ~35-50Å thick (depending upon the degree of inter-digitation). Second, the position of opposing platforms is quite variable, as large lateral and vertical displacements are present. This gives further support to our hypothesis that CARD-NBD linkers of Apaf-1 molecules may act as flexible tethers for the disk (Yuan et al., 2010
A CARD-CARD disk is present on the heptameric Apaf-1 platform
To test the idea of a flexible tether, we created a mutant Apaf-1 with a thrombin cleavage site inserted in the linker between the CARD and helix α8 (Apaf-1tb; ). Apoptosomes were assembled with Apaf-1tb and their mobility on a glycerol gradient was similar to wild type complexes (, lanes 6-8). When these complexes were treated with thrombin, CARDs were released en masse and remained at the top of the gradient (, lanes 1-3), while heptameric platforms started to aggregate and ran in fractions 9-12 (). These results are consistent with Apaf-1 CARDs being flexibly-arrayed on the apoptosome (), where they can interact with pc-9 CARDs. In addition, the CARD-NBD linker is disordered in the crystal structure of Apaf-1 (residues 1-591; Riedl et al., 2005
). Hence, it was not surprising that the thrombin site in the linker is accessible in Apaf-1 monomers ().
Apaf-1 CARDs are flexibly-linked to the central hub in the human apoptosome
To estimate the size of the disk, we visualized pc-9 CARD apoptosomes by rotary metal shadowing after freeze-fracture and etching (Heuser, 1989
; Supplemental Methods
). In top views, a strongly-contrasted, disk-like feature is visible above the platform (, white arrows). The disk had a diameter of ~100Å (after correcting for the metal coating) and was present in most particles. We also imaged ground state apoptosomes as a control. In these images, we observed a weakly contrasted ring above the hepatmeric platform. These rings (and partial rings) may represent flexibly-bound Apaf-1 CARDs that interact with each other to some extent (, white arrows). Based on these studies, we conclude that pc-9 and Apaf-1 CARDs form a novel disk that is flexibly-tethered to the central hub. Clearly, disk formation is a key step in activation because it would concentrate pc-9 catalytic domains in a region above the apoptosome to facilitate zymogen activation.
Role of the CARD-p20 linker in pc-9 activation
Procaspase-9 has a potential linker of ~69 residues between the CARD and p20 domain, although the actual linker may be comprised of the 49 residues inclusive of Asn92-Gly140 (; Renatus et al., 2001
). In a recent experiment, a 6 residue linker was sufficient to activate two pc-9 catalytic domains attached to a GCN4 leucine zipper (Yin et al., 2006
). Hence, the function of the much longer linker in pc-9 is mysterious. To study the role of the CARD-p20 linker, we made a series of truncations that removed 7, 20 and 30 residues directly downstream of an inserted thrombin site (). When factoring in extra residues from the thrombin site, these mutant procaspases have effective linker lengths of −1, −14 and −24 residues relative to wild type pc-9. A ribbon diagram of the pc-9 dimer is shown in Figure S3
with CARD-p20 linkers drawn roughly to scale for wild type and Δ30L molecules. We then measured the proteolytic activity of pc-9t linker mutants with Ac-LEHD-AFC, in the presence and absence of the apoptosome. For this experiment, we titrated the relative amount of each protein to give a pc-9 to Apaf-1 ratio of ~1:1 (see glycerol gradients, Figure S4
). Initial activity curves derived from the fluorescent product were obtained for complexes in a physiological buffer (PB) and in each case, we also measured baseline activities after cutting the pc-9t linker with thrombin to release the p20-p10 domains. Initial rates were normalized to the activity of the pc-9t apoptosome (100) and are plotted as a histogram in . These data provide three important observations.
The CARD-p20 linker plays a critical role in pc-9 activation
First, apoptosomes with pc-9t (+6 linker) showed a 50% reduction in their activity relative to complexes made with pc-9t Δ7L/−1. Thus, lengthening the wild type linker can lower activity. We suggest that the thrombin site my impair function of the CARD-p20 linker, perhaps by mediating local aggregation, which lowers activation by ~ 2.5-fold. Second, proteolytic activity was maximal for pc-9t Δ7L/−1, which has a linker that is nearly the same length as in wild type pc-9. Third, when the linker was shortened further (Δ20L/−14 and Δ30L/−24), proteolytic activity of the complexes dropped dramatically (~4 and 9-fold, respectively). To show the generality of our results, we also tested the activity of apoptosomes with wild type pc-9 molecules that contained a Δ30L/−30 mutation in the CARD-p20 linker. These experiments were done to eliminate possible side effects due to the thrombin site in the CARD-p20 linker and due to the D315A mutation in the p20-p10 linker. As shown in the next sections, each of these mutations may affect the overall activity level of pc-9 on the apoptosome. Based on fluorescence-based proteolysis assays, we found a significant defect in pc-9Δ30L apoptosomes relative to pc-9 complexes (). This shows that truncation of the CARD-p20 linker to ~19 residues leads to a significant loss of pc-9 activity on the apoptosome (~80%).
Procaspase-9 apoptosomes cleave and activate pc-3. We investigated this reaction using a pc-3 substrate in which the active site residue (Cys163) was mutated to alanine, to prevent self cleavage during bacterial over-expression. In this experiment, we used a 4-fold excess of pc-3 (C163A) relative to pc-9t and carried out a 30 min time course at 37°C for apoptosomes with pc-9t and with each of the 3 linker mutants. The cleavage of pc-3 to give p20 and p10 subunits was reduced significantly with pc-9tΔ24L and Δ30L apoptosomes, relative to complexes with pc-9t or pc-9tΔ7L (). In all cases, mock reactions showed no pc-3 cleavage (not shown). In addition, the overall activity level with pc-3 as a substrate paralleled the activity of these complexes with a small polypeptide substrate. We conclude that the defect in pc-9tΔ30L apoptosomes extends to the proteolytic processing of pc-3. In addition, apoptosomes with the pc-9tΔ7L mutant had the highest activity with either substrate. Not unexpectedly, these experiments suggest that the optimum linker length for activation is present in wild type pc-9 zymogens.
Next, we wondered if the activation defect in pc-9 molecules with a truncated linker might arise from an inability to assemble a CARD-CARD disk. To test this idea, we assembled apoptosomes with pc-9tΔ30L and released the catalytic domains with thrombin. We then froze these pc9 CARD apoptosomes, imaged the complexes and determined a structure at ~19Å resolution with EMAN2 (Tang et al., 2007
). A blurred disk was present in the final, c7 symmetrized 3D map (), which suggests that there is no gross defect in disk formation with this mutant.
Role of the p20-p10 linker
We observed a 25-30-fold activation of pc-9tΔ7L molecules on the apoptosome relative to the complexes treated with thrombin (). However, this value was lower than expected (Shi, 2004
). We reasoned that this may be due in part, to the D315A mutation in the p20-p10 linker. To test this idea, we measured the proteolytic activities of apoptosomes containing pc-9t with the D315A mutation and of complexes without this mutation (pc-9t wt). In order to assess the effect of the thrombin site, we also evaluated apoptosomes assembled with pc-9 (D315A) or pc-9 (). In panels 1 and 3, the D315A point mutation significantly reduced cleavage activity towards pc-3, relative to complexes in panels 2 and 4 that contained pc-9 molecules without this linker mutation. In addition, time courses with wild type pc9t and pc-9 showed extra density on the gels at t=0, due to the p35 and p10 subunits, which arise from bacterial cleavage of the p20-p10 linker. These bands overlapped the pc-3 and p10/cytochrome c bands, respectively (). The relative effects of the D315A and thrombin site mutants on pc-9 activation were also quantitated with the fluorescence-based proteolysis assay, as discussed later. We conclude that the D315A mutation in the p20-p10 linker has a significant effect on the final level of pc-9 activation (see next section for a possible explanation).
Properties of the pc-9 apoptosome
Procaspase-9 binding to the apoptosome
To evaluate the possible role of pc-9 binding in activation, we first compared the accessibility of the CARD-NBD linker in apoptosomes assembled with Apaf-1tb that contained either pc-9 or pc-9 (D315A). In this experiment, active complexes were treated with thrombin and purified on glycerol gradients. We found that clip sites in CARD-NBD linkers were more strongly protected when pc-9 was bound to the apoptosome (, left), relative to complexes with pc-9 (D315A) (, right). To more clearly visualize the protection, we probed the complexes with thrombin and then visualized them directly on a gel (). Since CARD-NBD linkers are located between the disk and the platform, the protection experiments suggested that pc-9 catalytic domains may bind to the apoptosome. If this is the case, then wild type catalytic domains of pc-9 may bind with a higher affinity than domains containing the D315A mutation.
Next, we asked if pc-9 catalytic domains could be visualized on the apoptosome. We froze apoptosomes with wild type pc-9 in LSB and determined a structure without symmetry restraints, using ~20,000 particles and EMAN2 (Tang et al., 2007
). To avoid bias, we started with an apoptosome that did not contain the CARD-CARD disk. After refinement, the 3D map revealed the p20 and p10 catalytic domains of a single pc-9 on the central hub, adjacent to the disk (). The FSC0.5
indicated a resolution of 16.9Å and edge-view classes revealed the disk and adjacent pc-9 density on the platform (Figures S5A, S5B
). Interestingly, helix α9 and strand β5 of an NBD are located in the “footprint” of the pc-9 catalytic domain, along with the NBD-HD1 linker (). In addition, bound pc-9 catalytic domains may also interact with helix α8 in the NBD of an adjacent subunit. However, a gap is present between the catalytic domains and the NBDs (see asterisk, ). This gap cannot be accounted for by the docked models, which suggests that part of the CARD-p20 linker may become ordered to help form the pc-9/NBD interface. This may explain why pc-9 apoptosomes with a Δ30L deletion in the zymogen are significantly less active than wild type complexes ().
Asymmetric structure of the pc-9 apoptosome
The precision of our current docking is limited by the resolution. In this case, an unambiguous choice cannot be made between the orientation in and a second position, defined by a 180 degree rotation about the long axis of the density. However, two aspects of pc-9 function are more consistent with the position shown in . First, the docking explains why a D315A mutation in the p20-p10 linker of pc-9 may down-regulate activation. In a pc-9 monomer, the uncleaved p20-p10 linker may interfere with formation of the NBD interface, because it would pass through the density footprint between the two components (Figure S6
and , middle and right panels). When Asp315 is cleaved, flexible L2 and L2′ ends of the p20-p10 linker may move laterally, allowing the catalytic domains to interact more efficiently with the hub. The p20-p10 loop also backtracks from Asp315 into the active site where it anchors the catalytically active Cys285 (Figure S6
, right). Thus, changes in this region induced by binding and auto-processing of the p20-p10 loop could activate the pc-9 monomer. Second, pc-9 is inhibited by the BIR3 domain of the X-inhibitor of apoptosis (XIAP), which forms a complex with the N-terminus (L2′) of the p10 subunit created by auto-processing (Shiozaki et al., 2003
). In our model, the BIR3-p10 complex would block activation by preventing the catalytic domains of pc-9 from binding to the hub. A higher resolution 3D map will be required to pin down the precise orientation of pc-9 catalytic domains on the hub.
In our 3D map, the CARD-CARD disk is located off-center relative to the central hub and appears to be tilted (Figures
, ). The acentric disk has apparent dimensions of ~80 × 80 × 35Å but the actual diameter of the disk as determined from rotary metal shadowing is ~100Å (see dashed circle in ). This is consistent with the idea that the disk is somewhat blurred in the 3D map because of flexible CARD-NBD linkers (Yuan et al., 2010
). We wondered if the disk may have been forced to move off-center when the catalytic domains of pc-9 are bound to the hub or alternatively, whether the disk may assemble in an acentric position relative to the hub. To evaluate these possibilities, we reprocessed a large dataset of pc-9 CARD apoptosomes from Yuan et al. (2010)
without symmetry restraints. These particles lack the pc-9 catalytic domain, yet the disk in the resulting 3D map is clearly acentric and tilted (), as it is in pc-9 apoptosomes (Figures
, ). Since the disk remains in an acentric position after catalytic domains have been released, it seems likely that co-assembly of pc-9 and Apaf-1 CARDs may create a mismatch between the disk and central hub. This also suggests that the disk itself may not have cylindrical symmetry.
Asymmetric structure of the pc-9 CARD apoptosome
In summary, the presence of a mismatch between the disk and central hub could pave the way for pc-9 catalytic domains to bind to a single site on the hub. This may occur because the acentric disk could block access to other binding sites, especially those on the far side of the hub relative to the observed binding site (). Since no additional density is present on adjacent Apaf-1 subunits, it seems that catalytic domains of only one pc-9 may be bound to this asymmetric proteolysis machine. We also suspect that binding may be a dynamic process with catalytic domains coming on and off the platform, because a single clip with thrombin released all catalytic domains from the pc-9t apoptosome, while the disk remained intact (Yuan et al., 2010
; next section).
Caspase-3 interactions with the apoptosome
The mechanism of pc-3 activation by the pc-9 apoptosome is not well understood. As a first step, we verified previous observations of caspase-3 binding to the apoptosome (Bratton et al., 2001a
; Hill et al., 2004
; Yin et al., 2006
; Malladi et al., 2009
). In this experiment, we added a 3-4 fold excess of unprocessed pc-3 (C163A) to pc-9t (D315A) apoptosomes in LSB and after 60 min the cleavage reaction was treated with thrombin. Proteins were then run on a glycerol gradient and visualized by SDS PAGE. Significant amounts of caspase-3 co-migrated with the pc-9 CARD apoptosome, as shown by the p20 subunit (note that the p10 subunit is overlapped with cytochrome c; , lanes 8-11). Released pc-9 catalytic domains, pc-3 and some caspase-3 stayed at the top of gradient. We then confirmed that pc-3 does not bind to the apoptosome. In this experiment, pc-9t apoptosomes were treated with thrombin to release catalytic domains, unprocessed pc-3 (C163A) was added for 60 min and the sample was run on a glycerol gradient. Under these conditions, no proteolysis of pc-3 occurred and most of the procaspase remained at the top of the gradient. While there was some streaking of pc-3 aggregates into the gradient (, lanes 1-5), these molecules were not associated with pc-9 CARD apoptosomes, which peaked in fractions 6-8. Hence, unprocessed pc-3 does not bind strongly to the apoptosome even though the zymogen is a constitutive dimer like caspase-3. These results are in line with previous data from Bratton et al. (2001a)
in which cell lysates were supplemented with recombinant caspases. When taken together, the data suggest that caspase-3 binding sites on the apoptosome are able to discriminate between active and inactive conformations of the caspase. In addition, caspase-3 is able to cleave Apaf-1 at Asp271 within the NBD (Bratton et al., 2001b
). In our apoptosome model, this cleavage site is buried within the interface between adjoining Apaf-1 subunits and therefore is unlikely to serve as a robust caspase-3 binding site (see Bratton et al., 2001a
; Hill et al., 2004
; Yin et al., 2006
Activation of pc-3 on the human apoptosome
We then investigated whether caspase-3 would compete successfully with wild type pc-9 for binding sites on the apoptosome. To test this idea, we assembled apoptosomes with wild type pc-9t. These complexes were run on a glycerol gradient or pre-treated with thrombin before the gradient step. In these experiments, wild type pc-9 catalytic domains were released from the apoptosome after thrombin treatment (, left and center panels). Hence, the affinity of wild type pc-9 catalytic domains for the apoptosome is greatly enhanced by formation of the CARD-CARD disk. We then incubated pc-9t wt apoptosomes with pc-3 (C163A) for 60 min, treated the complexes with thrombin and ran them on a glycerol gradient. This created caspase-3/pc-9 CARD apoptosomes that migrated into the gradient (, right panel), while released pc-9 catalytic domains remained at the top (not shown). Since caspase-3 dimers remain bound to the apoptosome when wild-type pc-9 catalytic domains are released by thrombin, the data suggest that caspase-3 dimers are bound to the central hub, rather than being bound by flexibly-tethered catalytic domains of pc-9.
To characterize these binding sites, we asked if pc-3 and caspase-3 would protect a thrombin site in CARD-NBD linkers of apoptosomes assembled with Apaf-1tb. We incubated pc-9 apoptosomes with pc-3 for either 10 min or 60 min at 37°C, before thrombin addition. Complexes were then run on glycerol gradients and visualized by SDS PAGE. As expected, we found that pc-3 did not protect the CARD-NBD linker from thrombin, since the zymogen is not strongly bound (, top left; fractions 9-11). However, caspase-3 was able to protect the linker (, top right; fractions 6-8). We then compared the ability of pc-9 and caspase-3 to protect CARD-NBD linkers in the apoptosome. For this experiment, we made appropriate complexes and probed them directly with thrombin. The degree of linker protection was much greater for caspase-3 than for pc-9 in these complexes and CARD-NBD linkers were more protected than in control apoptosomes (, bottom). When taken together, the data suggest that pc-9 and caspase-3 may use overlapping binding sites on the central hub, since they are both able to protect the CARD-NBD linker.
We also asked if pc-9t apoptosomes would proteolyze Ac-LEHD-AFC when caspase-3 was bound. In this experiment, we monitored pc-9 activity because caspase-3 (C163A) is catalytically inactive. The activity of pc-9t apoptosomes was normalized to 100 (, left). When pc-3 was added at t=0, the activity of pc-9t apoptosomes was diminished by ~30%. Note that the assay itself took ~15 min and some pc-3 was processed during this time. However, pc-9t activity in the caspase-3 complexes was reduced nearly to background levels when pc-3 was added 60 min before starting the assay (compare , left set). We also tested the effect of caspase-3 on apoptosomes with bound pc-9 molecules that lacked the D315A mutation, including complexes with pc-9t wt or pc-9 (, middle and right). In both cases, pc-9 molecules without the D315A mutation were significantly more active than pc-9t (D315A) molecules (), as shown previously (). Cleavage of unprocessed pc-3 to caspase-3 resulted in a dramatic loss of pc-9 activity to near background levels in both cases (). Based on our data, a possible explanation for these results is that pc-9 catalytic domains may be displaced from the central hub by caspase-3. This leads to a loss of pc-9 activity while the catalytic domains remain associated with the apoptosome through their linkers to the stable CARD-CARD disk.