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- PDB-5wve: Apaf-1-Caspase-9 holoenzyme -

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Basic information

Entry
Database: PDB / ID: 5wve
TitleApaf-1-Caspase-9 holoenzyme
Components
  • (Apoptotic protease-activating factor ...) x 2
  • Caspase
  • Cytochrome c
KeywordsAPOPTOSIS / apoptosis holoenzyme
Function / homology
Function and homology information


caspase-9 / response to G1 DNA damage checkpoint signaling / caspase complex / regulation of apoptotic DNA fragmentation / cytochrome c-heme linkage / Formation of apoptosome / apoptosome / cytochrome complex / leukocyte apoptotic process / glial cell apoptotic process ...caspase-9 / response to G1 DNA damage checkpoint signaling / caspase complex / regulation of apoptotic DNA fragmentation / cytochrome c-heme linkage / Formation of apoptosome / apoptosome / cytochrome complex / leukocyte apoptotic process / glial cell apoptotic process / cysteine-type endopeptidase activator activity / response to cobalt ion / Caspase activation via Dependence Receptors in the absence of ligand / Activation of caspases through apoptosome-mediated cleavage / Regulation of the apoptosome activity / SMAC (DIABLO) binds to IAPs / SMAC(DIABLO)-mediated dissociation of IAP:caspase complexes / fibroblast apoptotic process / AKT phosphorylates targets in the cytosol / mitochondrial electron transport, cytochrome c to oxygen / epithelial cell apoptotic process / cysteine-type endopeptidase activator activity involved in apoptotic process / platelet formation / response to anesthetic / mitochondrial electron transport, ubiquinol to cytochrome c / TP53 Regulates Transcription of Caspase Activators and Caspases / Constitutive Signaling by AKT1 E17K in Cancer / Transcriptional Regulation by E2F6 / forebrain development / intrinsic apoptotic signaling pathway in response to endoplasmic reticulum stress / signal transduction in response to DNA damage / positive regulation of execution phase of apoptosis / cellular response to dexamethasone stimulus / cellular response to transforming growth factor beta stimulus / heat shock protein binding / response to nutrient / cardiac muscle cell apoptotic process / intrinsic apoptotic signaling pathway / protein maturation / positive regulation of apoptotic signaling pathway / response to ischemia / NOD1/2 Signaling Pathway / neural tube closure / apoptotic signaling pathway / enzyme activator activity / kidney development / ADP binding / protein processing / mitochondrial intermembrane space / SH3 domain binding / intrinsic apoptotic signaling pathway in response to DNA damage / cellular response to UV / nervous system development / response to estradiol / peptidase activity / positive regulation of neuron apoptotic process / neuron apoptotic process / secretory granule lumen / regulation of apoptotic process / response to lipopolysaccharide / ficolin-1-rich granule lumen / cell differentiation / response to hypoxia / electron transfer activity / positive regulation of apoptotic process / cysteine-type endopeptidase activity / nucleotide binding / apoptotic process / heme binding / lipid binding / DNA damage response / Neutrophil degranulation / protein kinase binding / protein-containing complex / mitochondrion / proteolysis / extracellular exosome / extracellular region / ATP binding / metal ion binding / identical protein binding / nucleus / cytosol / cytoplasm
Similarity search - Function
CASP9, CARD domain / Apoptotic Protease-Activating Factor 1, CARD domain / : / Apoptotic protease-activating factor 1-like, winged-helix domain / Apoptotic protease-activating factor 1 / APAF-1 helical domain / APAF-1 helical domain / Apoptotic protease-activating factors, helical domain / Caspase recruitment domain / NB-ARC ...CASP9, CARD domain / Apoptotic Protease-Activating Factor 1, CARD domain / : / Apoptotic protease-activating factor 1-like, winged-helix domain / Apoptotic protease-activating factor 1 / APAF-1 helical domain / APAF-1 helical domain / Apoptotic protease-activating factors, helical domain / Caspase recruitment domain / NB-ARC / NB-ARC domain / Cytochrome c, class IA/ IB / CARD domain / CARD caspase recruitment domain profile. / Caspase recruitment domain / Peptidase C14 family / Peptidase family C14A, His active site / Caspase family histidine active site. / Peptidase C14, caspase non-catalytic subunit p10 / Peptidase family C14A, cysteine active site / Caspase family cysteine active site. / Caspase family p10 domain profile. / Peptidase C14A, caspase catalytic domain / Caspase, interleukin-1 beta converting enzyme (ICE) homologues / Peptidase C14, p20 domain / Caspase family p20 domain profile. / : / Caspase domain / Cytochrome c / Caspase-like domain superfamily / Cytochrome c family profile. / Cytochrome c-like domain / Cytochrome c-like domain superfamily / Death-like domain superfamily / WD domain, G-beta repeat / G-protein beta WD-40 repeat / WD40 repeat, conserved site / Trp-Asp (WD) repeats signature. / Trp-Asp (WD) repeats profile. / Trp-Asp (WD) repeats circular profile. / WD40 repeats / WD40 repeat / WD40-repeat-containing domain superfamily / Winged helix-like DNA-binding domain superfamily / WD40/YVTN repeat-like-containing domain superfamily / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
2'-DEOXYADENOSINE 5'-TRIPHOSPHATE / PROTOPORPHYRIN IX CONTAINING FE / Caspase-9 / Apoptotic protease-activating factor 1 / Cytochrome c / Caspase-9
Similarity search - Component
Biological speciesHomo sapiens (human)
Equus caballus (horse)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.4 Å
AuthorsLi, Y. / Zhou, M. / Hu, Q. / Shi, Y.
Funding support China, United Kingdom, 3items
OrganizationGrant numberCountry
Ministry of Science and Technology2014ZX09507003006 to YS China
National Natural Science Foundation of China(projects 31430020, 31130002, and 31321062 to YS China
UK Medical Research Council(MC_UP_A025_1013, to SHWS United Kingdom
CitationJournal: Proc Natl Acad Sci U S A / Year: 2017
Title: Mechanistic insights into caspase-9 activation by the structure of the apoptosome holoenzyme.
Authors: Yini Li / Mengying Zhou / Qi Hu / Xiao-Chen Bai / Weiyun Huang / Sjors H W Scheres / Yigong Shi /
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 ...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.
History
DepositionDec 24, 2016Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Feb 8, 2017Provider: repository / Type: Initial release
Revision 1.1Mar 1, 2017Group: Database references
Revision 1.2Nov 13, 2024Group: Data collection / Database references ...Data collection / Database references / Derived calculations / Structure summary
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / em_admin / em_image_scans / pdbx_entry_details / pdbx_modification_feature / struct_conn
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _em_admin.last_update / _struct_conn.pdbx_dist_value / _struct_conn.ptnr1_auth_asym_id / _struct_conn.ptnr1_auth_comp_id / _struct_conn.ptnr1_auth_seq_id / _struct_conn.ptnr1_label_asym_id / _struct_conn.ptnr1_label_atom_id / _struct_conn.ptnr1_label_comp_id / _struct_conn.ptnr1_label_seq_id / _struct_conn.ptnr2_auth_asym_id / _struct_conn.ptnr2_auth_comp_id / _struct_conn.ptnr2_auth_seq_id / _struct_conn.ptnr2_label_asym_id / _struct_conn.ptnr2_label_atom_id / _struct_conn.ptnr2_label_comp_id

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Structure visualization

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Assembly

Deposited unit
A: Apoptotic protease-activating factor 1
B: Cytochrome c
C: Apoptotic protease-activating factor 1
D: Cytochrome c
E: Apoptotic protease-activating factor 1
F: Cytochrome c
G: Apoptotic protease-activating factor 1
H: Cytochrome c
I: Apoptotic protease-activating factor 1
J: Cytochrome c
K: Apoptotic protease-activating factor 1
L: Cytochrome c
M: Apoptotic protease-activating factor 1
N: Cytochrome c
O: Apoptotic protease-activating factor 1
P: Apoptotic protease-activating factor 1
Q: Apoptotic protease-activating factor 1
R: Apoptotic protease-activating factor 1
S: Caspase
T: Caspase
U: Caspase
V: Caspase
W: Apoptotic protease-activating factor 1
X: Apoptotic protease-activating factor 1
Y: Caspase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)1,213,03046
Polymers1,205,10725
Non-polymers7,92421
Water00
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area62840 Å2
ΔGint-237 kcal/mol
Surface area430040 Å2

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Components

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Apoptotic protease-activating factor ... , 2 types, 13 molecules ACEGIKMOPQRWX

#1: Protein
Apoptotic protease-activating factor 1 / APAF-1


Mass: 142023.672 Da / Num. of mol.: 7
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: APAF1, KIAA0413
Production host: Insect cell expression vector pTIE1 (others)
References: UniProt: O14727
#3: Protein
Apoptotic protease-activating factor 1 / APAF-1


Mass: 11575.185 Da / Num. of mol.: 6 / Fragment: CARD domain, UNP residues 1-102
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: APAF1, KIAA0413
Production host: Insect cell expression vector pTIE1 (others)
References: UniProt: O14727

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Protein , 2 types, 12 molecules BDFHJLNSTUVY

#2: Protein
Cytochrome c


Mass: 11856.793 Da / Num. of mol.: 7
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Equus caballus (horse) / Gene: CYCS, CYC / Production host: Bacteria (eubacteria) / References: UniProt: P00004
#4: Protein
Caspase / caspase-9


Mass: 11698.449 Da / Num. of mol.: 5 / Fragment: CARD domain, UNP residues 1-100
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Production host: Bacteria (eubacteria) / References: UniProt: A8K7U6, UniProt: P55211*PLUS

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Non-polymers , 3 types, 21 molecules

#5: Chemical
ChemComp-DTP / 2'-DEOXYADENOSINE 5'-TRIPHOSPHATE


Mass: 491.182 Da / Num. of mol.: 7 / Source method: obtained synthetically / Formula: C10H16N5O12P3
#6: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 7 / Source method: obtained synthetically / Formula: Mg
#7: Chemical
ChemComp-HEM / PROTOPORPHYRIN IX CONTAINING FE / HEME


Mass: 616.487 Da / Num. of mol.: 7 / Source method: obtained synthetically / Formula: C34H32FeN4O4

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Details

Has protein modificationY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

Component
IDNameTypeEntity IDParent-IDSource
1Apaf-1 apoptosomeCOMPLEX#1-#40RECOMBINANT
2Apaf-1COMPLEX#11RECOMBINANT
3cytochrome cCOMPLEX#21RECOMBINANT
4Apaf-1 CARDCOMPLEX#31RECOMBINANT
5caspase-9 CARDCOMPLEX#41RECOMBINANT
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
11Homo sapiens (human)9606
21Equus caballus (horse)9796
31Homo sapiens (human)9606
41Homo sapiens (human)9606
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-IDPlasmid
11Insect cell expression vector pTIE1 (others)266783pFasBac
21Bacteria (eubacteria)2
31Insect cell expression vector pTIE1 (others)266783pFasBac
41Bacteria (eubacteria)2
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company
MicroscopyModel: FEI POLARA 300
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD
Image recordingElectron dose: 32 e/Å2 / Film or detector model: FEI FALCON II (4k x 4k)

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Processing

CTF correctionType: NONE
3D reconstructionResolution: 4.4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 240130 / Symmetry type: POINT

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