Changes in Apaf-1 conformation that drive apoptosome assembly.
ABSTRACT: Apoptosome assembly is highly regulated in the intrinsic cell death pathway. To better understand this step, we created an improved model of the human apoptosome using a crystal structure of full length Apaf-1 and a single particle, electron density map at ~9.5 Å resolution. The apoptosome model includes N-terminal domains of Apaf-1, cognate ?-propellers, and cytochrome c. A direct comparison of Apaf-1 in the apoptosome and as a monomer reveals conformational changes that occur during the first two steps of assembly. This includes an induced-fit mechanism for cytochrome c binding to regulatory ?-propellers, which is dependent on shape and charge complementarity, and a large rotation of the nucleotide binding module during nucleotide exchange. These linked conformational changes create an extended Apaf-1 monomer and drive apoptosome assembly. Moreover, the N-terminal CARD in the inactive Apaf-1 monomer is not shielded from other proteins by ?-propellers. Hence, the Apaf-1 CARD may be free to interact with a procaspase-9 CARD either before or during apoptosome assembly. Irrespective of the timing, the end product of assembly is a holo-apoptosome with an acentric CARD-CARD disk and tethered pc-9 catalytic domains. Subsequent activation of pc-9 leads to a proteolytic cascade and cell death.
Project description:Apaf-1 coassembles with cytochrome c to form the apoptosome, which then binds and activates procaspase-9 (pc-9). We removed pc-9 catalytic domains from the holoapoptosome by site-directed thrombinolysis. A structure of the resulting apoptosome-pc-9 CARD complex was then determined at approximately 9.5 A resolution. In our model, the central hub is constructed like other AAA+ protein rings but also contains novel features. At higher radius, the regulatory region of each Apaf-1 is comprised of tandem seven and eight blade beta-propellers with cytochrome c docked between them. Remarkably, Apaf-1 CARDs are disordered in the ground state. During activation, each Apaf-1 CARD interacts with a pc-9 CARD and these heterodimers form a flexibly tethered "disk" that sits above the central hub. When taken together, the data reveal conformational changes during Apaf-1 assembly that allow pc-9 activation. The model also provides a plausible explanation for the effects of NOD mutations that have been mapped onto the central hub.
Project description:In response to cell death signals, an active apoptosome is assembled from Apaf-1 and procaspase-9 (pc-9). Here we report a near atomic structure of the active human apoptosome determined by cryo-electron microscopy. The resulting model gives insights into cytochrome c binding, nucleotide exchange and conformational changes that drive assembly. During activation an acentric disk is formed on the central hub of the apoptosome. This disk contains four Apaf-1/pc-9 CARD pairs arranged in a shallow spiral with the fourth pc-9 CARD at lower occupancy. On average, Apaf-1 CARDs recruit 3 to 5 pc-9 molecules to the apoptosome and one catalytic domain may be parked on the hub, when an odd number of zymogens are bound. This suggests a stoichiometry of one or at most, two pc-9 dimers per active apoptosome. Thus, our structure provides a molecular framework to understand the role of the apoptosome in programmed cell death and disease.
Project description: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.
Project description:The cytosolic adaptor protein Apaf-1 is a key player in the intrinsic pathway of apoptosis. Binding of mitochondrially released cytochrome c and of dATP or ATP to Apaf-1 induces the formation of the heptameric apoptosome complex, which in turn activates procaspase-9. We have re-investigated the chain of events leading from monomeric autoinhibited Apaf-1 to the functional apoptosome in vitro. We demonstrate that Apaf-1 does not require energy from nucleotide hydrolysis to eventually form the apoptosome. Despite a low intrinsic hydrolytic activity of the autoinhibited Apaf-1 monomer, nucleotide hydrolysis does not occur at any stage of the process. Rather, mere binding of ATP in concert with the binding of cytochrome c primes Apaf-1 for assembly. Contradicting the current view, there is no strict requirement for an adenine base in the nucleotide. On the basis of our results, we present a new model for the mechanism of apoptosome assembly.
Project description:The apoptosome is a platform that activates apical procaspases in response to intrinsic cell death signals. Biochemical and structural studies in the past two decades have extended our understanding of apoptosome composition and structure, while illuminating the requirements for initiator procaspase activation. A number of studies have now provided high-resolution structures for apoptosomes from C. elegans (CED-4), D. melanogaster (Dark), and H. sapiens (Apaf-1), which define critical protein interfaces, including intra and interdomain interactions. This work also reveals interactions of apoptosomes with their respective initiator caspases, CED-3, Dronc and procaspase-9. Structures of the human apoptosome have defined the requirements for cytochrome c binding, which triggers the conversion of inactive Apaf-1 molecules to an extended, assembly competent state. While recent data have provided a detailed understanding of apoptosome formation and procaspase activation, they also highlight important evolutionary differences with functional implications for caspase activation. Comparison of the CARD/CARD disks and apoptosomes formed by CED-4, Dark and Apaf-1. Cartoons of the active states of the CARD-CARD disks, illustrating the two CED-4 CARD tetrameric ring layers (CED4a and CED4b; top row) and the binding of 8 Dronc CARDs and between 3-4 pc-9 CARDs, to the Dark and Apaf-1 CARD disk respectively (middle and lower rows). Ribbon diagrams of the active CED-4, Dark and Apaf-1 apoptosomes are shown (right column).
Project description: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 ?-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, ?-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 ?-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.
Project description:The apoptotic protease-activating factor 1 (Apaf-1) controls the onset of many known forms of intrinsic apoptosis in mammals. Apaf-1 exists in normal cells as an autoinhibited monomer. Upon binding to cytochrome c and dATP, Apaf-1 oligomerizes into a heptameric complex known as the apoptosome, which recruits and activates cell-killing caspases. Here we present an atomic structure of an intact mammalian apoptosome at 3.8 Å resolution, determined by single-particle, cryo-electron microscopy (cryo-EM). Structural analysis, together with structure-guided biochemical characterization, uncovered how cytochrome c releases the autoinhibition of Apaf-1 through specific interactions with the WD40 repeats. Structural comparison with autoinhibited Apaf-1 revealed how dATP binding triggers a set of conformational changes that results in the formation of the apoptosome. Together, these results constitute the molecular mechanism of cytochrome c- and dATP-mediated activation of Apaf-1.
Project description:Activation of procaspase-9 on the apoptosome is a pivotal step in the intrinsic cell death pathway. We now provide further evidence that caspase recruitment domains of pc-9 and Apaf-1 form a CARD-CARD disk that is flexibly tethered to the apoptosome. In addition, a 3D reconstruction of the pc-9 apoptosome was calculated without symmetry restraints. In this structure, p20 and p10 catalytic domains of a single pc-9 interact with nucleotide binding domains of adjacent Apaf-1 subunits. Together, disk assembly and pc-9 binding create an asymmetric proteolysis machine. We also show that CARD-p20 and p20-p10 linkers play important roles in pc-9 activation. Based on the data, we propose a proximity-induced association model for pc-9 activation on the apoptosome. We also show that pc-9 and caspase-3 have overlapping binding sites on the central hub. These binding sites may play a role in pc-3 activation and could allow the formation of hybrid apoptosomes with pc-9 and caspase-3 proteolytic activities.
Project description:Apaf-1-like molecules assemble into a ring-like platform known as the apoptosome. This cell death platform then activates procaspases in the intrinsic cell death pathway. In this review, crystal structures of Apaf-1 monomers and CED-4 dimers have been combined with apoptosome structures to provide insights into the assembly of cell death platforms in humans, nematodes, and flies. In humans, the caspase recognition domains (CARDs) of procaspase-9 and Apaf-1 interact with each other to form a CARD-CARD disk, which interacts with the platform to create an asymmetric proteolysis machine. The disk tethers multiple pc-9 catalytic domains to the platform to raise their local concentration, and this leads to zymogen activation. These findings have now set the stage for further studies of this critical activation process on the apoptosome.
Project description:Cdc6 is the bifunctional AAA+ ATPase that assembles prereplicative complexes on origins of replication and activates p21(CIP1)- or p27(KIP1)-bound Cdk2. During the G(1)-S transition, the Cdc6 gene essential for chromosomal replication is activated by the E2F transcriptional factor. Paradoxically, Apaf-1 encoding the central component of the apoptosome is also activated at the same time and by E2F. Consequently, genes for antipodal life and death are regulated in the same manner by the same transcriptional factor. Here we report a striking solution to this paradox. Besides performing prereplicative complex assembly and Cdk2 activation, Cdc6 obstructed apoptosome assembly by forming stable complexes very likely with a monomer of cytochrome c-activated Apaf-1 molecules. This function depended on its own ATPase domain but not on the cyclin-binding motif. In proliferating rodent fibroblasts, Cdc6 continued to block apoptosome assembly induced by a non-cytochrome c or some other mechanism, suppressing seemingly unintended apoptosis when promoting cell proliferation. Thus, Cdc6 is an AAA+ ATPase with three functions, all working for life.