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- PDB-7md3: The F1 region of apoptolidin-bound Saccharomyces cerevisiae ATP s... -

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Basic information

Entry
Database: PDB / ID: 7md3
TitleThe F1 region of apoptolidin-bound Saccharomyces cerevisiae ATP synthase
Components(ATP synthase subunit ...) x 4
KeywordsHYDROLASE/HYDROLASE INHIBITOR / F1-ATPase / inhibitor / anti-cancer / HYDROLASE / TRANSLOCASE / HYDROLASE-HYDROLASE INHIBITOR complex
Function / homology
Function and homology information


: / : / : / : / proton motive force-driven ATP synthesis / proton motive force-driven mitochondrial ATP synthesis / proton-transporting ATP synthase complex, catalytic core F(1) / H+-transporting two-sector ATPase / proton-transporting ATPase activity, rotational mechanism / proton-transporting ATP synthase activity, rotational mechanism ...: / : / : / : / proton motive force-driven ATP synthesis / proton motive force-driven mitochondrial ATP synthesis / proton-transporting ATP synthase complex, catalytic core F(1) / H+-transporting two-sector ATPase / proton-transporting ATPase activity, rotational mechanism / proton-transporting ATP synthase activity, rotational mechanism / ADP binding / mitochondrial inner membrane / ATP hydrolysis activity / mitochondrion / ATP binding
Similarity search - Function
ATP synthase, F1 complex, epsilon subunit, mitochondrial / ATP synthase, F1 complex, epsilon subunit superfamily, mitochondrial / Mitochondrial ATP synthase epsilon chain / ATP synthase, F1 complex, gamma subunit conserved site / ATP synthase gamma subunit signature. / ATP synthase, F1 complex, beta subunit / ATP synthase, alpha subunit, C-terminal domain superfamily / ATP synthase, F1 complex, gamma subunit / ATP synthase, F1 complex, gamma subunit superfamily / ATP synthase ...ATP synthase, F1 complex, epsilon subunit, mitochondrial / ATP synthase, F1 complex, epsilon subunit superfamily, mitochondrial / Mitochondrial ATP synthase epsilon chain / ATP synthase, F1 complex, gamma subunit conserved site / ATP synthase gamma subunit signature. / ATP synthase, F1 complex, beta subunit / ATP synthase, alpha subunit, C-terminal domain superfamily / ATP synthase, F1 complex, gamma subunit / ATP synthase, F1 complex, gamma subunit superfamily / ATP synthase / ATP synthase, alpha subunit, C-terminal / ATP synthase, F1 complex, alpha subunit / ATP synthase, F1 complex, alpha subunit nucleotide-binding domain / ATP synthase alpha/beta chain, C terminal domain / ATPase, F1/V1 complex, beta/alpha subunit, C-terminal / ATP synthase subunit alpha, N-terminal domain-like superfamily / ATPase, F1/V1/A1 complex, alpha/beta subunit, N-terminal domain superfamily / ATPase, F1/V1/A1 complex, alpha/beta subunit, N-terminal domain / ATP synthase alpha/beta family, beta-barrel domain / ATPase, alpha/beta subunit, nucleotide-binding domain, active site / ATP synthase alpha and beta subunits signature. / ATPase, F1/V1/A1 complex, alpha/beta subunit, nucleotide-binding domain / ATP synthase alpha/beta family, nucleotide-binding domain / ATPases associated with a variety of cellular activities / AAA+ ATPase domain / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
ADENOSINE-5'-TRIPHOSPHATE / PHOSPHATE ION / Chem-ZH7 / ATP synthase subunit beta / ATP synthase subunit gamma / ATP synthase subunit alpha / ATP synthase subunit epsilon, mitochondrial
Similarity search - Component
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.3 Å
AuthorsGuo, H. / Rubinstein, J.L.
Funding support Canada, United States, 8items
OrganizationGrant numberCountry
Canadian Institutes of Health Research (CIHR)PJT162186 Canada
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01 GM092218 United States
National Institutes of Health/National Cancer Institute (NIH/NCI)R01 CA226833 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35 GM133552 United States
National Institutes of Health/National Heart, Lung, and Blood Institute (NIH/NHLBI)K23 HL138291 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)T32 GM065086 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)T32 GM007347 United States
National Institutes of Health/National Cancer Institute (NIH/NCI)F30 CA236131 United States
CitationJournal: Nat Chem Biol / Year: 2022
Title: Apoptolidin family glycomacrolides target leukemia through inhibition of ATP synthase.
Authors: Benjamin J Reisman / Hui Guo / Haley E Ramsey / Madison T Wright / Bradley I Reinfeld / P Brent Ferrell / Gary A Sulikowski / W Kimryn Rathmell / Michael R Savona / Lars Plate / John L ...Authors: Benjamin J Reisman / Hui Guo / Haley E Ramsey / Madison T Wright / Bradley I Reinfeld / P Brent Ferrell / Gary A Sulikowski / W Kimryn Rathmell / Michael R Savona / Lars Plate / John L Rubinstein / Brian O Bachmann /
Abstract: Cancer cells have long been recognized to exhibit unique bioenergetic requirements. The apoptolidin family of glycomacrolides are distinguished by their selective cytotoxicity towards oncogene- ...Cancer cells have long been recognized to exhibit unique bioenergetic requirements. The apoptolidin family of glycomacrolides are distinguished by their selective cytotoxicity towards oncogene-transformed cells, yet their molecular mechanism remains uncertain. We used photoaffinity analogs of the apoptolidins to identify the F subcomplex of mitochondrial ATP synthase as the target of apoptolidin A. Cryogenic electron microscopy (cryo-EM) of apoptolidin and ammocidin-ATP synthase complexes revealed a novel shared mode of inhibition that was confirmed by deep mutational scanning of the binding interface to reveal resistance mutations which were confirmed using CRISPR-Cas9. Ammocidin A was found to suppress leukemia progression in vivo at doses that were tolerated with minimal toxicity. The combination of cellular, structural, mutagenesis, and in vivo evidence defines the mechanism of action of apoptolidin family glycomacrolides and establishes a path to address oxidative phosphorylation-dependent cancers.
History
DepositionApr 3, 2021Deposition site: RCSB / Processing site: RCSB
Revision 1.0Aug 11, 2021Provider: repository / Type: Initial release
Revision 1.1May 29, 2024Group: Data collection / Database references / Category: chem_comp_atom / chem_comp_bond / citation / Item: _citation.journal_id_ISSN

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Structure visualization

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Assembly

Deposited unit
A: ATP synthase subunit alpha
B: ATP synthase subunit alpha
C: ATP synthase subunit alpha
D: ATP synthase subunit beta
E: ATP synthase subunit beta
F: ATP synthase subunit beta
G: ATP synthase subunit gamma
H: ATP synthase subunit epsilon, mitochondrial
hetero molecules


Theoretical massNumber of molelcules
Total (without water)358,66016
Polymers355,8418
Non-polymers2,8198
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: microscopy
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area37020 Å2
ΔGint-207 kcal/mol
Surface area102330 Å2

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Components

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ATP synthase subunit ... , 4 types, 8 molecules ABCDEFGH

#1: Protein ATP synthase subunit alpha


Mass: 55007.402 Da / Num. of mol.: 3 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae (brewer's yeast) / Strain: USY006 / References: UniProt: A0A6A5Q4L9
#2: Protein ATP synthase subunit beta


Mass: 51181.082 Da / Num. of mol.: 3 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae (brewer's yeast) / Strain: USY006
References: UniProt: A0A6A5PX46, H+-transporting two-sector ATPase
#3: Protein ATP synthase subunit gamma


Mass: 30657.160 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae (brewer's yeast) / Strain: USY006 / References: UniProt: A0A6A5Q493
#4: Protein ATP synthase subunit epsilon, mitochondrial / ATPase subunit epsilon


Mass: 6618.359 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae (brewer's yeast) / Strain: USY006 / References: UniProt: P21306

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Non-polymers , 4 types, 8 molecules

#5: Chemical ChemComp-ATP / ADENOSINE-5'-TRIPHOSPHATE


Mass: 507.181 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C10H16N5O13P3 / Comment: ATP, energy-carrying molecule*YM
#6: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: Mg
#7: Chemical ChemComp-PO4 / PHOSPHATE ION


Mass: 94.971 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: PO4
#8: Chemical ChemComp-ZH7 / (3~{E},5~{E},7~{E},9~{R},10~{R},11~{E},13~{E},17~{S},18~{S},20~{S})-18-methoxy-20-[(~{R})-[(2~{R},3~{R},4~{S},5~{R},6~{R})-6-[(2~{R})-3-methoxy-2-[(2~{R},4~{S},5~{S},6~{S})-5-[(2~{S},4~{R},5~{R},6~{R})-4-methoxy-6-methyl-5-oxidanyl-oxan-2-yl]oxy-4,6-dimethyl-4-oxidanyl-oxan-2-yl]oxy-propyl]-3,5-dimethyl-2,4-bis(oxidanyl)oxan-2-yl]-oxidanyl-methyl]-10-[(2~{R},3~{S},4~{S},5~{R},6~{S})-5-methoxy-6-methyl-3,4-bis(oxidanyl)oxan-2-yl]oxy-3,5,7,9,13-pentamethyl-17-oxidanyl-1-oxacycloicosa-3,5,7,11,13-pentaen-2-one / Apoptolidin A


Mass: 1129.370 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C58H96O21 / Feature type: SUBJECT OF INVESTIGATION

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Details

Has ligand of interestY

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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

ComponentName: The F1 region of apoptolidin-bound Saccharomyces cerevisiae ATP synthase
Type: COMPLEX / Entity ID: #1-#4 / Source: NATURAL
Molecular weightValue: 0.4 MDa / Experimental value: NO
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / Strain: USY006
Buffer solutionpH: 7.4
SpecimenConc.: 15 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER/RHODIUM / Grid mesh size: 400 divisions/in. / Grid type: Homemade
VitrificationInstrument: FEI VITROBOT MARK III / Cryogen name: ETHANE-PROPANE / Humidity: 100 % / Chamber temperature: 4 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 75000 X / Calibrated magnification: 135922 X / Nominal defocus min: 1100 nm / Calibrated defocus max: 2500 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm / Alignment procedure: ZEMLIN TABLEAU
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 9.5 sec. / Electron dose: 43 e/Å2 / Film or detector model: FEI FALCON IV (4k x 4k) / Num. of real images: 4019
Image scansWidth: 4096 / Height: 4096

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Processing

Software
NameVersionClassification
phenix.real_space_refine1.17.1_3660refinement
PHENIX1.17.1_3660refinement
EM software
IDNameVersionCategory
2EPUimage acquisition
4cryoSPARC2.15CTF correction
9cryoSPARC2.15initial Euler assignment
10cryoSPARC2.15final Euler assignment
11cryoSPARC2.15classification
12cryoSPARC2.153D reconstruction
13cryoSPARC2.15model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1189085
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 477847 / Algorithm: BACK PROJECTION / Num. of class averages: 3 / Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL / Space: RECIPROCAL
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 50.72 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.006123396
ELECTRON MICROSCOPYf_angle_d0.6531830
ELECTRON MICROSCOPYf_chiral_restr0.04363856
ELECTRON MICROSCOPYf_plane_restr0.00394068
ELECTRON MICROSCOPYf_dihedral_angle_d13.28673420

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