title: Mechanistic insights into caspase-9 activation by the structure of the apoptosome holoenzyme.
abstract: Mammalian intrinsic apoptosis requires activation of the initiator caspase-9, which then cleaves and activates the effector caspases to execute cell killing. The heptameric Apaf-1 apoptosome is indispensable for caspase-9 activation by together forming a holoenzyme. The molecular mechanism of caspase-9 activation remains largely enigmatic. Here, we report the cryoelectron microscopy (cryo-EM) structure of an apoptotic holoenzyme and structure-guided biochemical analyses. The caspase recruitment domains (CARDs) of Apaf-1 and caspase-9 assemble in two different ways: a 4:4 complex docks onto the central hub of the apoptosome, and a 2:1 complex binds the periphery of the central hub. The interface between the CARD complex and the central hub is required for caspase-9 activation within the holoenzyme. Unexpectedly, the CARD of free caspase-9 strongly inhibits its proteolytic activity. These structural and biochemical findings demonstrate that the apoptosome activates caspase-9 at least in part through sequestration of the inhibitory CARD domain.
authors.affiliation: Beijing 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.
authors.forename: Yini
authors.initials: Y
authors.lastname: Li
authors.affiliation: Beijing 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.
authors.forename: Mengying
authors.initials: M
authors.lastname: Zhou
authors.affiliation: Beijing 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.
authors.forename: Qi
authors.initials: Q
authors.lastname: Hu
authors.affiliation: Medical Research Council Laboratory of Molecular Biology, Cambridge Biomedical Campus, Cambridge CB2 0QH, United Kingdom.
authors.forename: Xiao-Chen
authors.initials: XC
authors.lastname: Bai
authors.affiliation: Beijing 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.
authors.forename: Weiyun
authors.initials: W
authors.lastname: Huang
authors.affiliation: Medical Research Council Laboratory of Molecular Biology, Cambridge Biomedical Campus, Cambridge CB2 0QH, United Kingdom.
authors.forename: Sjors H W
authors.identifier: 0000-0002-0462-6540
authors.initials: SH
authors.lastname: Scheres
authors.affiliation: Beijing 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; shi-lab@tsinghua.edu.cn.
authors.forename: Yigong
authors.initials: Y
authors.lastname: Shi
mesh_terms: D017209:Apoptosis; D053478:Apoptosomes / Q000737:chemistry / Q000378:metabolism* / Q000648:ultrastructure; D053477:Apoptotic Protease-Activating Factor 1 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D053453:Caspase 9 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000071476:Caspase Activation and Recruitment Domain / Q000235:genetics; D020285:Cryoelectron Microscopy; D004789:Enzyme Activation; D020035:Holoenzymes / Q000737:chemistry / Q000378:metabolism* / Q000648:ultrastructure; D006801:Humans; D008958:Models, Molecular; D009154:Mutation; D011485:Protein Binding; D000072417:Protein Domains; D055503:Protein Multimerization
chemical_list: C506592:APAF1 protein, human; D053478:Apoptosomes; D053477:Apoptotic Protease-Activating Factor 1; D020035:Holoenzymes; D053453:Caspase 9
keywords: apoptosome; caspase activation; caspase-9; cryo-EM; holoenzyme