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

Yini Lia,1, Mengying Zhoua,1,QiHu(胡奇)a,1, Xiao-chen Baib, Weiyun Huanga, Sjors H. W. Scheresb, and Yigong Shi (施一公)a,2

aBeijing Advanced Innovation Center for Structural Biology, Tsinghua-Peking Center for Life Sciences, School of Life Sciences and School of Medicine, Tsinghua University, Beijing 100084, China; and bMedical Research Council Laboratory of Molecular Biology, Cambridge Biomedical Campus, Cambridge CB2 0QH, United Kingdom

Contributed by Yigong Shi, December 17, 2016 (sent for review December 5, 2016; reviewed by Emad S. Alnemri and Hao Wu) Mammalian intrinsic requires activation of the initiator The 9.5-Å cryoelectron microscopy (cryo-EM) structure of an caspase-9, which then cleaves and activates the effector apoptosome–caspase-9 complex revealed some EM density lobes to execute cell killing. The heptameric Apaf-1 apoptosome is in- that are flexibly attached to the center as well as the periphery of dispensable for caspase-9 activation by together forming a holoen- the holoenzyme (14, 15). An unspecified number of the CARD zyme. The molecular mechanism of caspase-9 activation remains domains and a single molecule of the caspase-9 protease domain largely enigmatic. Here, we report the cryoelectron microscopy were thought to be located at the center and the periphery, re- (cryo-EM) structure of an apoptotic holoenzyme and structure- spectively. Although this information might be crucial for un- guided biochemical analyses. The caspase recruitment domains derstanding caspase-9 activation, neither the identities of the (CARDs) of Apaf-1 and caspase-9 assemble in two different ways: CARD domains nor their interactions with the apoptosome were a 4:4 complex docks onto the central hub of the apoptosome, and identified. The cryo-EM structure of the apoptosome at a near- a 2:1 complex binds the periphery of the central hub. The interface atomic resolution reveals how the monomeric Apaf-1 assembles between the CARD complex and the central hub is required for caspase-9 activation within the holoenzyme. Unexpectedly, the into a functional heptameric apoptosome, but the CARD domains CARD of free caspase-9 strongly inhibits its proteolytic activity. exhibit no obvious EM density (16).

These structural and biochemical findings demonstrate that the In this manuscript, we report the cryo-EM structure of an intact BIOCHEMISTRY – apoptosome activates caspase-9 at least in part through sequestra- mammalian apoptosome caspase-9 holoenzyme at a markedly tion of the inhibitory CARD domain. improved resolution and systematic structure-guided biochemical analyses, which together provide important mechanistic insights apoptosome | caspase-9 | holoenzyme | caspase activation | cryo-EM into the activation of caspase-9 by the Apaf-1 apoptosome. Results poptosis, also called , plays an es- Asential role in the development of multicellular organisms EM of the Apoptosome Holoenzyme. An intact holoenzyme was and the maintenance of tissue homeostasis (1, 2). Apoptosis is assembled by incubating the full-length human Apaf-1 (residues – c executed by a number of sequentially activated caspases (3). 1 1,248) with excess amounts of equine cytochrome (CytC) and Caspases are divided into two classes: initiators and effectors (3). All caspases are synthesized as inactive zymogens in cells and Significance must undergo proteolytic cleavages to become fully activated. An effector caspase is cleaved and activated by an upstream initiator As a prototypical initiator caspase, caspase-9 has been rigor- caspase and is responsible for the cleavage of numerous cellular ously studied for 20 years. Although activation of the caspase-9 . An initiator caspase undergoes autocatalytic activation, zymogen and catalytic activity of the mature caspase-9 both which depends on a specific multimeric adaptor complex. strictly depend on the Apaf-1 apoptosome, the underlying In most known forms of intrinsic apoptosis, caspase-9 and mechanism remains poorly understood. Previous mechanistic studies relied heavily on engineered caspase-9 in the absence caspase-3/7 are the initiator and the downstream effector cas- of the Apaf-1 apoptosome. In this study, we tackle the problem pases, respectively. The multimeric protein complex required for through structure determination of the Apaf-1 apoptosome caspase-9 activation is the assembled heptameric Apaf-1 apopto- bound to caspase-9 and biochemical analysis of caspase-9 ac- some (4). The activation mechanisms of the initiator and effector tivity in the presence of the Apaf-1 apoptosome. The results caspases are quite different. The effector caspases, exemplified by demonstrate that the Apaf-1 apoptosome activates caspase-9 caspase-3, exist as stable homodimers. The activation of caspase-3, by two means: suppressing the inhibition mediated by the usually mediated by activated caspase-9, entails a single proteolytic caspase recruitment domains (CARD) domain and stimulating cleavage between the large and small subunits of caspase-3 (5). In the catalytic activity of the protease domain. contrast, caspase-9 exists as a monomeric, inactive zymogen in cells. The intrachain cleavage is insufficient for the activation of Author contributions: Y.L., M.Z., Q.H., and Y.S. designed research; Y.L., M.Z., Q.H., X.-c.B., caspase-9 (6–8). Caspase-9 undergoes autocatalytic cleavages on W.H., and S.H.W.S. performed research; Y.L., M.Z., Q.H., X.-c.B., W.H., S.H.W.S., and Y.S. analyzed data; and Y.L., M.Z., and Y.S. wrote the paper. binding to the apoptosome, but the cleaved caspase-9 is only cat- Reviewers: E.S.A., Kimmel Cancer Institute; and H.W., Boston Children’s Hospital and alytically activated when bound to the apoptosome, prompting the Harvard Medical School. – concept of a caspase-9 holoenzyme (9 11). Despite decades of The authors declare no conflict of interest. intense investigation, it remains largely unknown how the oligo- Freely available online through the PNAS open access option. meric apoptosome facilitates caspase-9 activation. Most previous Data deposition: The atomic coordinates have been deposited in the Protein Data Bank, studies on caspase-9 activation used artificially engineered caspase-9 www.pdb.org (PDB ID 5WVE). and were conducted in the absence of the full-length Apaf-1 1Y.L., M.Z., and Q.H. contributed equally to this work. apoptosome. These studies show that homodimerization drives 2To whom correspondence should be addressed. Email: [email protected]. caspase-9 activation, and thus suggest a similar mechanism by the This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. apoptosome (12, 13). 1073/pnas.1620626114/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1620626114 PNAS Early Edition | 1of6 Downloaded by guest on September 23, 2021 human caspase-9 in the presence of 1 mM dATP. The assembled holoenzyme exhibited excellent solution behavior, as judged by gel filtration (Fig. S1A). The assembled holoenzyme was imaged under cryo-conditions on an FEI Tecnai Polara microscope, and 2,579 micrographs were collected (Fig. S1B); 401,784 particles were autopicked for reference-free 2D classification (Fig. S1C). After 3D classification, a subset of 240,130 particles was used for image construction, with different regions of the holoenzyme exhibiting vastly different resolution limits (Fig. S2). A lobe of compact globular density, present in nearly all particles and similar to that reported previously (14, 15), is lo- cated right above the center of the apoptosome (Figs. S2 and S3). An additional but smaller lobe of sickle-shaped density, present in about 10% of the particles, is found at the edge of the central hub, close to the WD40 repeats. The local resolution in these regions was truncated to ∼18 Å in the published structure of the apoptosome holoenzyme (14, 15). In our structure, applica- tion of local masking strategy for the heptameric apoptosome platform, the globular density above the central hub, and the sickle- shaped density at the edge of the central hub led to improved overall resolutions of 4.4, 5.1, and 6.9 Å, respectively (Fig. S3). The central hub of the apoptosome holoenzyme displays an identical structure as that reported previously (16).

Identification of the CARD Domains. Apaf-1 sequentially contains a CARD at the N terminus, a nucleotide-binding oligomerization domain, and two β-propellers of seven and eight WD40 repeats at the C terminus. The nucleotide-binding oligomerization do- main comprises a nucleotide-binding domain (NBD), a small helical domain, a winged-helix domain, and a large helical do- main. Caspase-9 consists of an N-terminal CARD and a protease domain. As previously observed (16), the central hub and the spokes of the apoptosome platform are formed by the nucleotide- Fig. 1. Cryo-EM structure of a mammalian apoptosome–caspase-9 holoen- binding oligomerization domains and the WD40 repeats, respec- zyme. (A) Two perpendicular views of the overall structure. Four ApCARD tively (Fig. 1A). Eight CARDs are identified in the globular domains and four C9CARD domains above the central hub are colored green density above the central hub, including a layer of four Apaf-1 and orange, respectively. (B) Structure of the heterotrimeric CARD complex CARDs (ApCARDs) docking onto the central hub and a second between two ApCARDs (green) and one C9CARD (orange) (17). Type I and layer of four caspase-9 CARDs (C9CARDs) on top of the type II interfaces are indicated between ApCARD and C9CARD. The six α-helices in each CARD are labeled H1–H6. (C) The central CARD complex can ApCARDs (Fig. 1A). be viewed as four overlapping heterotrimeric CARD complexes, each be- The assignment of the central CARD complex was based on tween two ApCARDs (green) and one C9CARD (orange). For example, C1 (a the observation that two ApCARDs and one C9CARD form a C9CARD) forms a heterotrimeric complex with A1 and A2 (two ApCARDs), C2 stable heterotrimeric complex through both type I and type II with A2 and A3, C3 with A3 and A4, and C4 with A4 and A1. (D) The CARD interfaces (17) (Fig. 1B). Helices H2 and H3 of ApCARD in- complex at the periphery of the central hub is a heterotrimeric CARD com- teract with helices H1 and H4 of C9CARD in the type I interface plex between two ApCARDs (green) and one C9CARD (orange). Notably, (18), whereas the intervening turns between H1 and H2 and about 15% of the holoenzymes have the peripheral CARD complex. This between H5 and H6 of ApCARD stack against the turns be- figure and Fig. 2 were prepared in PyMol (32). tween H2 and H3 and between H4 and H5 of C9CARD in the type II interface. The centrally located hetero-octameric CARD only three of the four ApCARDs directly contact the apoptosome complex, measuring 75 Å in diameter and 60 Å in height, can be platform (Fig. 2A). The 5.1-Å resolution is insufficient for accu- viewed as four overlapping heterotrimeric complexes (Fig. 1C). rate assignment of specific interactions involving amino acid side For three of the four C9CARDs in the central CARD complex, chains in the CARD complex. Despite this caveat, the close each interacts with two neighboring ApCARDs through type I and type II interfaces (Fig. 1C). The fourth C9CARD associates proximity of the secondary structural elements between ApCARDs with ApCARD through a type II interface only. Because both and the central hub allows modeling of potential interactions, which types of interface are known to be stable (17), assembly of the may guide experimental scrutiny of their functional importance. central CARD complex in this fashion maximizes the number of The interactions between ApCARDs and the central hub of type I and type II interfaces, and hence is thermodynamically the apoptosome are asymmetric. For two ApCARDs, each uses most favorable. its N-terminal helix H1 and the C-terminal portion of helix H4 to At 6.9 Å resolution, the sickle-shaped density at the edge of interact with surface regions of the NBD (Fig. 2B). Despite the central hub allowed identification and docking of two variations in the detailed interactions, the surface regions of the ApCARDs and one C9CARD, which are in perfect registry with NBD contacted by these two ApCARDs are similar. In one case, the reported heterotrimeric complex (17) (Fig. 1D). Notably, this the N-terminal residues Asp2 and Arg6 may hydrogen bond density was described as a single protease domain of caspase-9 in (H-bond) to Thr112 and Glu116, whereas Lys58 and Lys62 are in the previous holoenzyme structures (14, 15), as well as in a re- close proximity of Gln208 and Asp209 (Fig. 2C, Middle and cent report (19). Right). In the other case, Lys4 may donate H-bonds to Tyr109 and Glu175 (Fig. 2C, Left). The third ApCARD is placed further Interactions Involving the CARD Domains. In the central CARD away from the central hub and may only make limited contacts to complex, four C9CARDs are located on the upper layer, and the central hub.

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1620626114 Li et al. Downloaded by guest on September 23, 2021 Fig. 2. Interactions between the CARD complex and the central hub of the apoptosome platform. (A) The central CARD complex binds to the central hub through three discrete interfaces (boxed by three colored rectangles). (B) A close-up view on the interface that appears to have denser interactions than the other two interfaces. Two boxed regions are zoomed in to visualize the detailed interactions in C.(C) Three zoom- in views of the putative interactions at the interface between the CARD complex and the central hub. The indicated interactions are highly speculative because the resolution of the local EM density map does not allow determination of amino acid side chains.

The 6.9-Å resolution makes it challenging even to speculate on (Fig. 3B). In contrast to the controls, the variants M7 and M8 only potential interactions at the edge of the central hub. In the stimulated caspase-9 activity to ∼45–50% of the level by WT Apaf-

heterotrimeric CARD complex, the N-terminal H1 helix of one 1. Consistent with results of the other assay (Fig. 3A), the variants BIOCHEMISTRY ApCARD is in close proximity to an N-terminal helix of the M4, M5, and M6 exhibited severely compromised abilities to NBD (Fig. S4A). Polar and/or charged residues on H1, such as stimulate caspase-9 activity (Fig. 3B). These results demonstrate Arg6 and Asn7, and those on NBD, such as Gln137, Gln138, and that interactions between the central CARD complex and the Ser141, may interact with each other through H-bonds (Fig. central hub are essential for proper activation of caspase-9. S4B). Similarly, polar and/or charged amino acids from the Mutations in these Apaf-1 variants were designed to selec- N termini of helices H1 and H4 in the other ApCARD, including tively affect the interface between the central CARD complex Glu14, Lys18, and Gln50, may contact surface residues in WD1 and the central hub, but not the assembly of the apoptosome. To (Fig. S4C). confirm the design, we individually incubated five Apaf-1 vari- ants with CytC in the presence of dATP and applied the mixture Essential Role of the Central CARD Complex. To examine whether to gel filtration (Fig. 3C). These variants include two controls the interactions between the CARD complex and the central hub (M1 and M3) and three loss-of-function variants (M4, M5, and are functionally important, we introduced specific missense M6). All five Apaf-1 variants retained the same ability as WT mutations into Apaf-1 that were designed to weaken these in- Apaf-1 to assemble into the apoptosomes (Fig. 3C). teractions but have no effect on formation of the apoptosome. Why is the interface between the central CARD complex and We generated six such Apaf-1 variants, designated M3 through the central hub required for caspase-9 activation? One possibility M8, and two control variants, M1 (S141W, K142W, and K144W) is that compromised interactions may affect the integrity of the and M2 (L173W), that are predicted to have no effect on these apoptosome–caspase-9 holoenzyme. To examine this scenario, interactions (Fig. 3). These Apaf-1 variants were individually we assessed formation of a stable holoenzyme on gel filtration by incubated with caspase-9 in the presence of CytC and dATP; the incubating the assembled apoptosome variants with caspase-9. proteolytic activity of caspase-9 was examined using its physio- As anticipated, caspase-9 formed a stable holoenzyme complex logical substrate caspase-3 (C163A) (Fig. 3A). Free caspase-9 with WT Apaf-1 and the two control variants M1 and M3, as exhibited a low level of the proteolytic activity, which was dras- judged by comigration of the caspase-9 large subunit with the tically increased by the WT Apaf-1 apoptosome (lanes 5 and 6). apoptosome (Fig. 3D). In contrast, caspase-9 failed to comigrate As anticipated, the control variants M1 and M2 stimulated with the apoptosome assembled by each of the three Apaf-1 caspase-9 activity to the same level as WT Apaf-1 (lanes 7 and 8). variants M4, M5, and M6. These results suggest that the reduced The M3 variant, in which the mutation D209A targets an amino ability by these Apaf-1 variants to activate caspase-9 is likely acid at the interface between the CARD complex and the central attributable to a compromised ability to form the apoptosome hub, also functioned similarly as WT Apaf-1 (lane 9). This ob- holoenzyme. servation suggests that a single-point mutation may be insufficient for destabilization of the interface. Supporting this analysis, three An Inhibitory Role by Caspase-9 CARD. Unlike the effector caspases, variants, M4 (D209K and S211K), M5 (Q208K, D209K, and caspase-9 contains an N-terminal CARD that is required for its S211K), and M6 (K58E and K62E), each involving two or more activation. The recruitment of caspase-9 by the Apaf-1 apopto- mutations at the interface, led to markedly decreased caspase-9 some strictly depends on their respective CARD domains (18, activity compared with WT Apaf-1 (Fig. 3A,lanes10–12). 20). Two CARD complexes, one above the central hub and the The Apaf-1 variants M7 (D2R, K4E) and M8 (Y109W, T112W, other at the periphery, are prominently featured in the assem- and E116R) contain two and three mutations, respectively, at the bled apoptosome holoenzyme. We suspected a regulatory role by interface, yet both variants appeared to stimulate caspase-9 ac- C9CARD in addition to its known function in holoenzyme as- tivity to a similar level as WT Apaf-1 (Fig. 3A, lanes 13 and 14). To sembly. Indeed, the isolated protease domain of caspase-9 was better characterize these variants, we used a more sensitive cleav- previously found to exhibit a higher level of proteolytic activity age assay using the fluorogenic peptide substrate Ac-LEHD-AFC compared with the full-length caspase-9 (12). To investigate this

Li et al. PNAS Early Edition | 3of6 Downloaded by guest on September 23, 2021 Fig. 3. The interface between the CARD complex and the central hub of the apoptosome platform is essential for caspase-9 activation. (A) Mutations in Apaf-1 that compromise the interface led to de- creased caspase-9 activity. The representative SDS/ PAGE gels shown here contain results of the caspase-9 activity assays. Eight Apaf-1 variants, labeled M1–M8, are used in the assays, and their identities are tabu- lated below the gels. (B) Examination of caspase-9 activity using the fluorogenic peptide Ac-LEHD-AFC. (C) The Apaf-1 mutations exhibited no effect on for- mation of the apoptosome. The Apaf-1 variants were individually examined for apoptosome assembly by gel filtration (Left), and the peak fractions were visu- alized by SDS/PAGE and Coomassie staining (Right). (D) Apaf-1 mutations that led to reduced caspase-9 activity also compromised formation of the holoen- zyme. Three Apaf-1 variants (M4, M5, and M6) were individually examined for their abilities to assemble into a holoenzyme with WT caspase-9 by gel filtration (Left), and the peak fractions were visualized (Right).

scenario, we generated two caspase-9 variants: residues 98–416, higher than that of WT caspase-9 in the peptide-based assay (Fig. which no longer contains the C9CARD, and residues 140–416, 5C), consistent with an inhibitory role of C9CARD in caspase- which only contains the protease domain (Fig. 4A). Compared 9 homodimerization. with full-length caspase-9, the CARD-deleted variant (residues Intriguingly, compared with the full-length DC9, removal of 98–416) exhibited a markedly increased proteolytic activity to- the CARD in DC9 (residues 98–416) led to a 2.5-fold increase of ward the physiological substrate caspase-3 (C163A) (Fig. 4A, the proteolytic activity, and further truncation of the linker se- lanes 6 and 7). Removal of the linker sequences in caspase-9 led quences preceding the protease domain resulted in an additional to an even higher level of activity (lane 8). Similar results were threefold increase (Fig. 5C). We speculate that DC9 exists in an obtained using the fluorogenic peptide substrate Ac-LEHD- equilibrium between a large population of homodimers and a AFC, where removal of the CARD increased caspase-9 activity small population of monomers, and the presence of C9CARD by at least 10-fold (Fig. 4B). These results demonstrate that the and the ensuing linker sequences inhibit homodimerization. This CARD domain of caspase-9 directly inhibits its proteolytic ac- tivity. This finding strongly argues that assembly of the CARD speculation appears to be supported by the appearances of the complex in the apoptosome holoenzyme may serve to relieve the peaks for the various DC9 variants on gel filtration (Fig. 5A). inhibitory effect of C9CARD. Instead of the proposed inhibition of caspase-9 homodimerization, How does the CARD inhibit the proteolytic activity of the caspase-9 protease domain? One possibility is that, similar to effector caspases, free caspase-9 only has robust proteolytic ac- tivity in its homodimeric form, and C9CARD inhibits homo- dimerization. This hypothesis is particularly attractive because caspase-9 inhibition by the BIR3 domain of XIAP is mediated through heterodimerization that involves the homodimerization interface of caspase-9 (21). To investigate this scenario, we engineered three constitutively homodimeric caspase-9 variants using an established strategy, in which a five-residue β-strand at the homodimeric interface of caspase-3 was grafted onto cas- pase-9 (22). The resulting caspase-9 variants, hereafter referred to as DC9, remain mainly as stable homodimers on gel filtration Fig. 4. The CARD and the linker sequences preceding the protease domain (Fig. 5A). Compared with WT caspase-9, the full-length DC9 of caspase-9 directly inhibit the catalytic activity of caspase-9. (A) Removal of the CARD and the linker sequences preceding the caspase-9 protease do- exhibited a much higher level of proteolytic activity toward the main led to markedly increased catalytic activities compared with the full- physiological substrate caspase-3 (C163A) (Fig. 5B, lanes 7 and length, WT caspase-9. The physiological substrate caspase-3 (C163A) was 8) or the fluorogenic peptide Ac-LEHD-AFC (Fig. 5C). In used in the assays. (B) The catalytic activities of the caspase-9 variants were particular, the proteolytic activity of the full-length DC9 is ∼12-fold examined using the fluorogenic substrate Ac-LEHD-AFC.

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1620626114 Li et al. Downloaded by guest on September 23, 2021 BIOCHEMISTRY

Fig. 5. CARD-deleted, constitutive homodimeric caspase-9 exhibits a similar level of catalytic activity as the apoptosome–caspase-9 holoenzyme. (A) The engineered caspase-9 with its β6 strand replaced by that of caspase-3 was eluted from gel filtration as a stable homodimer. This is shown for the full-length caspase-9 (DC9), CARD-deleted (residues 98–416), and the protease domain alone (residues 140–416). (B) The protease domain of the constitutive homodi- meric caspase-9 (residues 140–416) exhibits a similar level of catalytic activity as the apoptosome caspase-9 holoenzyme. The physiological substrate caspase-3 (C163A) was used in the assays. (C) The catalytic activities of the caspase-9 variants were examined using the fluorogenic substrate Ac-LEHD-AFC.

C9CARD may directly inhibit the proteolytic activity through periphery of the central hub was identified to be a 2:1 complex binding to the active site or another region of caspase-9. The between ApCARD and C9CARD. This represents a major above experimental results fail to rule out this possibility. structural difference. Incubation of the full-length DC9 with the Apaf-1 apoptosome In this study, we demonstrate that the interface between the yielded a sixfold increase of the proteolytic activity. In fact, DC9 central CARD complex and the central hub of the apoptosome (residues 140–416) displayed a higher proteolytic activity toward platform is essential for caspase-9 activation. Mutations in Apaf-1 the substrate Ac-LEHD-AFC than the apoptosome–caspase-9 that compromise this interface had no effect on apoptosome as- holoenzyme (Fig. 5C). Intriguingly, the proteolytic activity of sembly but crippled caspase-9 recruitment and holoenzyme for- DC9 (residues 140–416) is slightly higher than that of the full- mation (Fig. 3). Both the proteolytic assays and the gel filtration length DC9 in the presence of the Apaf-1 apoptosome (Fig. 5 B runs were performed with a molar ratio of 5:1 for Apaf-1 over and C). This finding demonstrates that the homodimerized pro- caspase-9; the under-stoichiometric amount of caspase-9 proved tease domain of caspase-9 has a proteolytic activity that is at least important for maximizing the difference between WT and mutant comparable to or higher than that of the apoptosome caspase-9 Apaf-1 variants. holoenzyme. Given that the isolated ApCARD stably interacts with C9CARD (18), why would mutations not affecting the ApCARD- Discussion C9CARD interactions compromise caspase-9 recruitment into the During our manuscript preparation, we noted publication of a apoptosome? One explanation is that ApCARD in the assembled cryo-EM structure of the apoptosome–caspase-9 holoenzyme at apoptosome is unavailable to interact with C9CARD alone, and considerably improved resolutions over previous studies (19). caspase-9 recruitment is coupled to assembly of the octameric The average local resolutions for the apoptosome platform and CARD complex and its docking onto the central hub. In other the globular density above the central hub were reported to be words, assembly of the CARD complex and its interaction with 4.1 and 5.8 Å, respectively (19). The central globular density was the central hub are coupled, and thus both are required to allow assigned to a 4:4 complex between ApCARD and C9CARD with caspase-9 recruitment. Under this scenario, compromised inter- an identical arrangement as observed in this study. The density face between the CARD complex and the central hub no longer lobe at the periphery of the central hub, previously reported allows assembly of the CARD complex or recruitment of caspase-9. to be 18 Å resolution (14, 15), was again assigned to a single Why are only four ApCARDs involved in the central CARD caspase-9 protease domain (19). In our cryo-EM reconstruction, complex? The other three ApCARDs from the apoptosome cannot the average local resolutions for the apoptosome platform, the be stacked on top of the 4:4 CARD complex because the length of central globular density, and the sickle-shaped density at the the linker sequence between the CARD and the NBD (14 residues) periphery were estimated to be 4.4, 5.0, and 7.0 Å, respectively. is too short. These resolution limits allowed us to determine the identity of How does the assembly of the central CARD complex assist individual domains and their relative orientations. Different caspase-9 activation? We identified the inhibitory role of the from the published study (19), the sickle-shaped density at the CARD in controlling caspase-9 activity (Fig. 4). The CARD

Li et al. PNAS Early Edition | 5of6 Downloaded by guest on September 23, 2021 suppresses the proteolytic activity of caspase-9, and the linker Materials and Methods sequence preceding the protease domain further augments the Protein Purification and Holoenzyme Assembly. Human Apaf-1 and WT suppression. Thus, at least one compelling reason for assembly of caspase-9 were purified as described (16). The holoenzyme was assembled the central CARD complex is to move the CARD and the linker in the presence of 1 mM dATP by incubating Apaf-1, equine CytC, and sequences away from the protease domain of caspase-9. Despite caspase-9 at a molar ratio of 1:2:2. The dimeric caspase-9 was generated the removal of the CARD and the linker sequences, the catalytic as described (22). activity of caspase-9 (residues 140–416) is still only a fraction of that by WT caspase-9 in the presence of the apoptosome. Thus, EM. Cryo-EM images were recorded manually on an FEI Tecnai Polara electron the apoptosome must have another role in the activation of microscope operating at 300 kV. The 26 movie frames of each micrograph caspase-9. Elucidation of this role may require structural infor- were aligned by whole-image motion correction (23). Contrast transfer mation on the protease domain of caspase-9 in the holoenzyme. function parameters were estimated by CTFFIND4 (24), and 2D and 3D Unfortunately, the protease domain of caspase-9 remains invis- classifications were carried out using RELION (version 1.4-alpha) (25). The ible in the EM density maps. This could be because of either its particle-polishing approach was applied (26). Reported resolutions are based intrinsic property (no interaction with the apoptosome platform on Fourier Shell Correlation (FSC) curves between independently refined half-maps, using the 0.143 criterion (27). The resulting maps from refinement or the CARD complex) or disruption of such transient interac- were postprocessed by RELION and sharpened by a negative B-factor (28). tions involving the protease domain. In the former scenario, the Local resolution variations were estimated using ResMap (29). The structures protease domain of caspase-9 is left with literally only one of Apaf-1 apoptosome (PDB code 3JBT) (16) and 2:1 ApCARD-C9CARD complex choice: activation through homodimerization. In the latter sce- (PDB code 4RHW) (17) were docked into the overall maps by COOT (30) and nario, how to capture the transient interactions remains a fitted into density by CHIMERA (31). daunting task that, given the intensity of unsuccessful structural investigations, may have to be addressed by single-molecule bio- Analysis of Interactions Between Apaf-1 and Caspase-9. For apoptosome as- physical studies. sembly, Apaf-1 mutants were individually incubated with equine CytC at a In this study, we show that an engineered, constitutively molar ratio of 1:2 in the presence of 1 mM dATP. For holoenzyme assembly, homodimeric caspase-9 exhibits robust catalytic activity (Fig. 5 the apoptosome mutants were individually incubated with WT caspase-9 at a A–C). The catalytic activity of DC9 is significantly higher than molar ratio of 4:1. that in a previous report (22). This difference is likely a result of the different temperatures under which caspase-9 activity is ex- Caspase-9 Activity Assay. The effect of Apaf-1 mutations on caspase-9 activity amined. DC9 exhibits excellent solution behavior at 22 °C (assay was measured using caspase-3 (C163A) and Ac-LEHD-AFC. The molar ration temperature in this study) but is prone to precipitation at 37 °C between Apaf-1 and caspase-9 is 5:1. The assay was performed as described [assay temperature of the previous study (22)]. Remarkably, with (8). The basal activities of caspase-9 truncations were also measured as de- deletion of the CARD and the linker sequences, DC9 (residues scribed (8). 140–416) has a higher catalytic activity than the apoptosome– caspase-9 holoenzyme. Thus, it is possible that, in addition to re- ACKNOWLEDGMENTS. This work was supported by funds from Ministry of Science and Technology Grant 2014ZX09507003006 (to Y.S.); National Nat- lieving the inhibition of C9CARD, apoptosome serves to promote ural Science Foundation of China Projects 31430020, 31130002, and 31321062 dimerization of the protease domain of caspase-9. Scrutiny of this (to Y.S.); European Union Marie Curie Fellowship (X.-c.B,); and UK Medical hypothesis awaits further experimental evidence. Research Council Grant MC_UP_A025_1013 (to S.H.W.S.).

1. Horvitz HR (2003) Worms, life, and death (Nobel lecture). ChemBioChem 4(8): 17. Hu Q, et al. (2014) Molecular determinants of caspase-9 activation by the Apaf-1 697–711. apoptosome. Proc Natl Acad Sci USA 111(46):16254–16261. 2. Danial NN, Korsmeyer SJ (2004) Cell death: Critical control points. Cell 116(2):205–219. 18. Qin H, et al. (1999) Structural basis of procaspase-9 recruitment by the apoptotic 3. Shi Y (2002) Mechanisms of caspase activation and inhibition during apoptosis. Mol protease-activating factor 1. Nature 399(6736):549–557. Cell 9(3):459–470. 19. Cheng TC, Hong C, Akey IV, Yuan S, Akey CW (2016) A near atomic structure of the 4. Chai J, Shi Y (2014) Apoptosome and : Conserved machineries for active human apoptosome. eLife 5:5. caspase activation. Natl Sci Rev 1(1):101–118. 20. Li P, et al. (1997) Cytochrome c and dATP-dependent formation of Apaf-1/caspase-9 5. Riedl SJ, Shi Y (2004) Molecular mechanisms of caspase regulation during apoptosis. complex initiates an apoptotic protease cascade. Cell 91(4):479–489. Nat Rev Mol Cell Biol 5(11):897–907. 21. Shiozaki EN, et al. (2003) Mechanism of XIAP-mediated inhibition of caspase-9. Mol 6. Stennicke HR, et al. (1999) Caspase-9 can be activated without proteolytic processing. Cell 11(2):519–527. J Biol Chem 274(13):8359–8362. 22. Chao Y, et al. (2005) Engineering a dimeric caspase-9: A re-evaluation of the induced 7. Srinivasula SM, et al. (2001) A conserved XIAP-interaction motif in caspase-9 and proximity model for caspase activation. PLoS Biol 3(6):e183. Smac/DIABLO regulates caspase activity and apoptosis. Nature 410(6824):112–116. 23. Li X, et al. (2013) Electron counting and beam-induced motion correction enable near- 8. Hu Q, Wu D, Chen W, Yan Z, Shi Y (2013) Proteolytic processing of the caspase-9 atomic-resolution single-particle cryo-EM. Nat Methods 10(6):584–590. zymogen is required for apoptosome-mediated activation of caspase-9. J Biol Chem 24. Mindell JA, Grigorieff N (2003) Accurate determination of local defocus and specimen 288(21):15142–15147. tilt in electron microscopy. J Struct Biol 142(3):334–347. 9. Rodriguez J, Lazebnik Y (1999) Caspase-9 and APAF-1 form an active holoenzyme. 25. Scheres SH (2012) RELION: Implementation of a Bayesian approach to cryo-EM Genes Dev 13(24):3179–3184. structure determination. J Struct Biol 180(3):519–530. 10. Zou H, Li Y, Liu X, Wang X (1999) An APAF-1.cytochrome c multimeric complex is 26. Scheres SH (2014) Beam-induced motion correction for sub-megadalton cryo-EM a functional apoptosome that activates procaspase-9. JBiolChem274(17): particles. eLife 3:e03665. 11549–11556. 27. Chen S, et al. (2013) High-resolution noise substitution to measure overfitting and 11. Saleh A, Srinivasula SM, Acharya S, Fishel R, Alnemri ES (1999) Cytochrome c and validate resolution in 3D structure determination by single particle electron cry- dATP-mediated oligomerization of Apaf-1 is a prerequisite for procaspase-9 activa- omicroscopy. Ultramicroscopy 135:24–35. tion. J Biol Chem 274(25):17941–17945. 28. Rosenthal PB, Henderson R (2003) Optimal determination of particle orientation, 12. Boatright KM, et al. (2003) A unified model for apical caspase activation. Mol Cell absolute hand, and contrast loss in single-particle electron cryomicroscopy. J Mol Biol 11(2):529–541. 333(4):721–745. 13. Pop C, Timmer J, Sperandio S, Salvesen GS (2006) The apoptosome activates caspase-9 29. Kucukelbir A, Sigworth FJ, Tagare HD (2014) Quantifying the local resolution of cryo- by dimerization. Mol Cell 22(2):269–275. EM density maps. Nat Methods 11(1):63–65. 14. Yuan S, et al. (2010) Structure of an apoptosome-procaspase-9 CARD complex. 30. Emsley P, Cowtan K (2004) Coot: Model-building tools for molecular graphics. Acta Structure 18(5):571–583. Crystallogr D Biol Crystallogr 60(Pt 12 Pt 1):2126–2132. 15. Yuan S, et al. (2011) The holo-apoptosome: Activation of procaspase-9 and interac- 31. Pettersen EF, et al. (2004) UCSF Chimera–a visualization system for exploratory re- tions with caspase-3. Structure 19(8):1084–1096. search and analysis. J Comput Chem 25(13):1605–1612. 16. Zhou M, et al. (2015) Atomic structure of the apoptosome: Mechanism of cytochrome 32. DeLano WL (2002) The PyMOL Molecular Graphics System. www.pymol.org (Schrö- c- and dATP-mediated activation of Apaf-1. Genes Dev 29(22):2349–2361. dinger, Cambridge, MA).

6of6 | www.pnas.org/cgi/doi/10.1073/pnas.1620626114 Li et al. Downloaded by guest on September 23, 2021