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- PDB-7a8p: Structure of human mitochondrial RNA polymerase in complex with I... -

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Basic information

Entry
Database: PDB / ID: 7a8p
TitleStructure of human mitochondrial RNA polymerase in complex with IMT inhibitor.
ComponentsDNA-directed RNA polymerase, mitochondrial
KeywordsTRANSCRIPTION / Mitochondria / Polymerase / Inhibitor
Function / homology
Function and homology information


Mitochondrial transcription initiation / mitochondrial DNA-directed RNA polymerase complex / mitochondrial promoter sequence-specific DNA binding / transcription initiation at mitochondrial promoter / mitochondrial transcription / DNA primase activity / mitochondrial nucleoid / Transcriptional activation of mitochondrial biogenesis / DNA-directed 5'-3' RNA polymerase activity / DNA-directed RNA polymerase ...Mitochondrial transcription initiation / mitochondrial DNA-directed RNA polymerase complex / mitochondrial promoter sequence-specific DNA binding / transcription initiation at mitochondrial promoter / mitochondrial transcription / DNA primase activity / mitochondrial nucleoid / Transcriptional activation of mitochondrial biogenesis / DNA-directed 5'-3' RNA polymerase activity / DNA-directed RNA polymerase / 3'-5'-RNA exonuclease activity / sequence-specific DNA binding / mitochondrial matrix / protein-containing complex / mitochondrion / RNA binding
Similarity search - Function
DNA-directed RNA polymerase, N-terminal / DNA-directed RNA polymerase, N-terminal domain superfamily / DNA-directed RNA polymerase N-terminal / Bacteriophage-type RNA polymerase family active site signature 1. / DNA-directed RNA polymerase N-terminal / DNA-directed RNA polymerase, phage-type / : / DNA-dependent RNA polymerase / Bacteriophage-type RNA polymerase family active site signature 2. / Tetratricopeptide-like helical domain superfamily / DNA/RNA polymerase superfamily
Similarity search - Domain/homology
Chem-R4Q / DNA-directed RNA polymerase, mitochondrial
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.5 Å
AuthorsHillen, H.S. / Bonekamp, N. / Peter, B. / Felser, A. / Bergbrede, T. / Choidas, A. / Horn, M. / Unger, A. / di Lucrezia, R. / Atanassov, I. ...Hillen, H.S. / Bonekamp, N. / Peter, B. / Felser, A. / Bergbrede, T. / Choidas, A. / Horn, M. / Unger, A. / di Lucrezia, R. / Atanassov, I. / Li, X. / Koch, U. / Menninger, S. / Boros, J. / Habenberger, P. / Giavalisco, P. / Cramer, P. / Denzel, M. / Nussbaumer, P. / Klebl, B. / Falkenberg, M. / Gustafsson, C.M. / Larsson, N.G.
Funding support Sweden, Germany, 15items
OrganizationGrant numberCountry
Swedish Research Council2015-00418 Sweden
Swedish Research Council2013-3621 Sweden
Swedish Research Council2012-2583 Sweden
Knut and Alice Wallenberg Foundation Sweden
German Research Foundation (DFG)SFB1218/A06 Germany
European Research Council (ERC)Advanced Grant 2016-741366 Sweden
Swedish Research CouncilALFGBG-727491 Sweden
Swedish Research CouncilALFGBG-728151 Sweden
Swedish Research CouncilSLL2018.0471 Sweden
German Research Foundation (DFG)SFB860 Germany
German Research Foundation (DFG)SPP1935 Germany
European Research Council (ERC)693023 Germany
Volkswagen Foundation Germany
German Research Foundation (DFG)EXC 2067/1- 390729940 Germany
German Research Foundation (DFG)FOR2848 Germany
CitationJournal: Nature / Year: 2020
Title: Small-molecule inhibitors of human mitochondrial DNA transcription.
Authors: Nina A Bonekamp / Bradley Peter / Hauke S Hillen / Andrea Felser / Tim Bergbrede / Axel Choidas / Moritz Horn / Anke Unger / Raffaella Di Lucrezia / Ilian Atanassov / Xinping Li / Uwe Koch / ...Authors: Nina A Bonekamp / Bradley Peter / Hauke S Hillen / Andrea Felser / Tim Bergbrede / Axel Choidas / Moritz Horn / Anke Unger / Raffaella Di Lucrezia / Ilian Atanassov / Xinping Li / Uwe Koch / Sascha Menninger / Joanna Boros / Peter Habenberger / Patrick Giavalisco / Patrick Cramer / Martin S Denzel / Peter Nussbaumer / Bert Klebl / Maria Falkenberg / Claes M Gustafsson / Nils-Göran Larsson /
Abstract: Altered expression of mitochondrial DNA (mtDNA) occurs in ageing and a range of human pathologies (for example, inborn errors of metabolism, neurodegeneration and cancer). Here we describe first-in- ...Altered expression of mitochondrial DNA (mtDNA) occurs in ageing and a range of human pathologies (for example, inborn errors of metabolism, neurodegeneration and cancer). Here we describe first-in-class specific inhibitors of mitochondrial transcription (IMTs) that target the human mitochondrial RNA polymerase (POLRMT), which is essential for biogenesis of the oxidative phosphorylation (OXPHOS) system. The IMTs efficiently impair mtDNA transcription in a reconstituted recombinant system and cause a dose-dependent inhibition of mtDNA expression and OXPHOS in cell lines. To verify the cellular target, we performed exome sequencing of mutagenized cells and identified a cluster of amino acid substitutions in POLRMT that cause resistance to IMTs. We obtained a cryo-electron microscopy (cryo-EM) structure of POLRMT bound to an IMT, which further defined the allosteric binding site near the active centre cleft of POLRMT. The growth of cancer cells and the persistence of therapy-resistant cancer stem cells has previously been reported to depend on OXPHOS, and we therefore investigated whether IMTs have anti-tumour effects. Four weeks of oral treatment with an IMT is well-tolerated in mice and does not cause OXPHOS dysfunction or toxicity in normal tissues, despite inducing a strong anti-tumour response in xenografts of human cancer cells. In summary, IMTs provide a potent and specific chemical biology tool to study the role of mtDNA expression in physiology and disease.
History
DepositionAug 30, 2020Deposition site: PDBE / Processing site: PDBE
Revision 1.0Dec 30, 2020Provider: repository / Type: Initial release
Revision 1.1Jan 6, 2021Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation_author.identifier_ORCID
Revision 1.2May 1, 2024Group: Data collection / Database references / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / em_3d_fitting_list / pdbx_initial_refinement_model
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type

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Structure visualization

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Assembly

Deposited unit
A: DNA-directed RNA polymerase, mitochondrial
hetero molecules


Theoretical massNumber of molelcules
Total (without water)130,2772
Polymers129,8031
Non-polymers4741
Water00
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area0 Å2
ΔGint0 kcal/mol
Surface area40550 Å2
MethodPISA

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Components

#1: Protein DNA-directed RNA polymerase, mitochondrial / MtRPOL


Mass: 129803.094 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: POLRMT / Plasmid: ProEx-His6TEVD104mtRNAP / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: O00411, DNA-directed RNA polymerase
#2: Chemical ChemComp-R4Q / (3~{R})-1-[(2~{R})-2-[4-(2-chloranyl-4-fluoranyl-phenyl)-2-oxidanylidene-chromen-7-yl]oxypropanoyl]piperidine-3-carboxylic acid


Mass: 473.878 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C24H21ClFNO6 / Feature type: SUBJECT OF INVESTIGATION
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: Human POLRMT (lacking residues 1-104) in complex with IMT inhibitor.
Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 0.128 MDa / Experimental value: NO
Source (natural)Organism: Homo sapiens (human) / Organelle: Mitochondria
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria) / Strain: BL 21 (DE3) RIL / Plasmid: ProEx-His6TEVD104mtRNAP
Buffer solutionpH: 8
Buffer component
IDConc.NameBuffer-ID
120 mMTris/HCl1
2300 mMNaCl1
35 mMMgCl21
41 mMDTT1
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid type: UltrAuFoil R2/2
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277.15 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS / Details: Stage was tilted 40 deg.
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: SPOT SCAN
Electron lensMode: BRIGHT FIELD / Nominal magnification: 105000 X / Nominal defocus max: 3000 nm / Nominal defocus min: 500 nm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 36.25 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV

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Processing

EM software
IDNameVersionCategory
1Warpparticle selection
2SerialEMimage acquisition
4WarpCTF correction
7Coot0.9model fitting
9PHENIXmodel refinement
10cryoSPARCinitial Euler assignment
11RELION3.0.7final Euler assignment
12RELION3.0.7classification
13RELION3.0.73D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1446981
3D reconstructionResolution: 3.5 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 193651 / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: OTHER / Space: REAL
Atomic model buildingPDB-ID: 4BOC

4boc


Pdb chain-ID: A / Accession code: 4BOC / Source name: PDB / Type: experimental model

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