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- EMDB-11679: Structure of human mitochondrial RNA polymerase in complex with I... -

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Basic information

Entry
Database: EMDB / ID: EMD-11679
TitleStructure of human mitochondrial RNA polymerase in complex with IMT inhibitor.
Map dataPost-processed main map.
Sample
  • Complex: Human POLRMT (lacking residues 1-104) in complex with IMT inhibitor.
    • Protein or peptide: DNA-directed RNA polymerase, mitochondrial
  • Ligand: (3~{R})-1-[(2~{R})-2-[4-(2-chloranyl-4-fluoranyl-phenyl)-2-oxidanylidene-chromen-7-yl]oxypropanoyl]piperidine-3-carboxylic acid
KeywordsMitochondria / Transcription / 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
DNA-directed RNA polymerase, mitochondrial
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.5 Å
AuthorsHillen HS / Bonekamp N
Funding support Sweden, Germany, 15 items
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, 2020-
Header (metadata) releaseDec 30, 2020-
Map releaseDec 30, 2020-
UpdateMay 1, 2024-
Current statusMay 1, 2024Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.02
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.02
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7a8p
  • Surface level: 0.02
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_11679.png

Downloads & links

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Map

FileDownload / File: emd_11679.map.gz / Format: CCP4 / Size: 52.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationPost-processed main map.
Voxel sizeX=Y=Z: 0.834 Å
Density
Contour LevelBy AUTHOR: 0.02 / Movie #1: 0.02
Minimum - Maximum-0.08984552 - 0.13858746
Average (Standard dev.)0.00014561192 (±0.0028840627)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions240240240
Spacing240240240
CellA=B=C: 200.16 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.8340.8340.834
M x/y/z240240240
origin x/y/z0.0000.0000.000
length x/y/z200.160200.160200.160
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS240240240
D min/max/mean-0.0900.1390.000

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Supplemental data

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Mask #1

Fileemd_11679_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Refinement half-map 1.

Fileemd_11679_half_map_1.map
AnnotationRefinement half-map 1.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Refinement half-map 2.

Fileemd_11679_half_map_2.map
AnnotationRefinement half-map 2.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Human POLRMT (lacking residues 1-104) in complex with IMT inhibitor.

EntireName: Human POLRMT (lacking residues 1-104) in complex with IMT inhibitor.
Components
  • Complex: Human POLRMT (lacking residues 1-104) in complex with IMT inhibitor.
    • Protein or peptide: DNA-directed RNA polymerase, mitochondrial
  • Ligand: (3~{R})-1-[(2~{R})-2-[4-(2-chloranyl-4-fluoranyl-phenyl)-2-oxidanylidene-chromen-7-yl]oxypropanoyl]piperidine-3-carboxylic acid

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Supramolecule #1: Human POLRMT (lacking residues 1-104) in complex with IMT inhibitor.

SupramoleculeName: Human POLRMT (lacking residues 1-104) in complex with IMT inhibitor.
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Homo sapiens (human) / Organelle: Mitochondria
Molecular weightTheoretical: 128 KDa

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Macromolecule #1: DNA-directed RNA polymerase, mitochondrial

MacromoleculeName: DNA-directed RNA polymerase, mitochondrial / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO / EC number: DNA-directed RNA polymerase
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 129.803094 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MGHHHHHHEN LYFQSNARKV QMGAKDATPV PCGRWAKILE KDKRTQQMRM QRLKAKLQMP FQSGEFKALT RRLQVEPRLL SKQMAGCLE DCTRQAPESP WEEQLARLLQ EAPGKLSLDV EQAPSGQHSQ AQLSGQQQRL LAFFKCCLLT DQLPLAHHLL V VHHGQRQK ...String:
MGHHHHHHEN LYFQSNARKV QMGAKDATPV PCGRWAKILE KDKRTQQMRM QRLKAKLQMP FQSGEFKALT RRLQVEPRLL SKQMAGCLE DCTRQAPESP WEEQLARLLQ EAPGKLSLDV EQAPSGQHSQ AQLSGQQQRL LAFFKCCLLT DQLPLAHHLL V VHHGQRQK RKLLTLDMYN AVMLGWARQG AFKELVYVLF MVKDAGLTPD LLSYAAALQC MGRQDQDAGT IERCLEQMSQ EG LKLQALF TAVLLSEEDR ATVLKAVHKV KPTFSLPPQL PPPVNTSKLL RDVYAKDGRV SYPKLHLPLK TLQCLFEKQL HME LASRVC VVSVEKPTLP SKEVKHARKT LKTLRDQWEK ALCRALRETK NRLEREVYEG RFSLYPFLCL LDEREVVRML LQVL QALPA QGESFTTLAR ELSARTFSRH VVQRQRVSGQ VQALQNHYRK YLCLLASDAE VPEPCLPRQY WEALGAPEAL REQPW PLPV QMELGKLLAE MLVQATQMPC SLDKPHRSSR LVPVLYHVYS FRNVQQIGIL KPHPAYVQLL EKAAEPTLTF EAVDVP MLC PPLPWTSPHS GAFLLSPTKL MRTVEGATQH QELLETCPPT ALHGALDALT QLGNCAWRVN GRVLDLVLQL FQAKGCP QL GVPAPPSEAP QPPEAHLPHS AAPARKAELR RELAHCQKVA REMHSLRAEA LYRLSLAQHL RDRVFWLPHN MDFRGRTY P CPPHFNHLGS DVARALLEFA QGRPLGPHGL DWLKIHLVNL TGLKKREPLR KRLAFAEEVM DDILDSADQP LTGRKWWMG AEEPWQTLAC CMEVANAVRA SDPAAYVSHL PVHQDGSCNG LQHYAALGRD SVGAASVNLE PSDVPQDVYS GVAAQVEVFR RQDAQRGMR VAQVLEGFIT RKVVKQTVMT VVYGVTRYGG RLQIEKRLRE LSDFPQEFVW EASHYLVRQV FKSLQEMFSG T RAIQHWLT ESARLISHMG SVVEWVTPLG VPVIQPYRLD SKVKQIGGGI QSITYTHNGD ISRKPNTRKQ KNGFPPNFIH SL DSSHMML TALHCYRKGL TFVSVHDCYW THAADVSVMN QVCREQFVRL HSEPILQDLS RFLVKRFCSE PQKILEASQL KET LQAVPK PGAFDLEQVK RSTYFFS

UniProtKB: DNA-directed RNA polymerase, mitochondrial

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Macromolecule #2: (3~{R})-1-[(2~{R})-2-[4-(2-chloranyl-4-fluoranyl-phenyl)-2-oxidan...

MacromoleculeName: (3~{R})-1-[(2~{R})-2-[4-(2-chloranyl-4-fluoranyl-phenyl)-2-oxidanylidene-chromen-7-yl]oxypropanoyl]piperidine-3-carboxylic acid
type: ligand / ID: 2 / Number of copies: 1 / Formula: R4Q
Molecular weightTheoretical: 473.878 Da
Chemical component information

ChemComp-R4Q:
(3~{R})-1-[(2~{R})-2-[4-(2-chloranyl-4-fluoranyl-phenyl)-2-oxidanylidene-chromen-7-yl]oxypropanoyl]piperidine-3-carboxylic acid

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 8
Component:
ConcentrationName
20.0 mMTris/HCl
300.0 mMNaCl
5.0 mMMgCl2
1.0 mMDTT
GridModel: UltrAuFoil R2/2 / Material: GOLD / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
DetailsStage was tilted 40 deg.
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 36.25 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELD / Nominal defocus max: 3.0 µm / Nominal defocus min: 0.5 µm / Nominal magnification: 105000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 1446981
Startup modelType of model: INSILICO MODEL
Final reconstructionNumber classes used: 1 / Resolution.type: BY AUTHOR / Resolution: 3.5 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0.7) / Number images used: 193651
Initial angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: cryoSPARC
Final angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: RELION (ver. 3.0.7)
Final 3D classificationNumber classes: 4 / Avg.num./class: 100000 / Software - Name: RELION (ver. 3.0.7)
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

4boc


Chain - Chain ID: A / Chain - Source name: PDB / Chain - Initial model type: experimental model
RefinementSpace: REAL / Protocol: OTHER
Output model

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

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