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- EMDB-16294: Cryo-EM structure of nanodisc-reconstituted wildtype human MRP4 (... -

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Basic information

Entry
Database: EMDB / ID: EMD-16294
TitleCryo-EM structure of nanodisc-reconstituted wildtype human MRP4 (in complex with topotecan)
Map dataCryo-EM map for nanodisc-reconstituted wildtype human MRP4 in complex with TPT
Sample
  • Organelle or cellular component: Cryo-EM structure of nanodisc-reconstituted wildtype human MRP4 (in complex with topotecan)
    • Protein or peptide: ATP-binding cassette sub-family C member 4
  • Ligand: (S)-10-[(DIMETHYLAMINO)METHYL]-4-ETHYL-4,9-DIHYDROXY-1H-PYRANO[3',4':6,7]INOLIZINO[1,2-B]-QUINOLINE-3,14(4H,12H)-DIONE
KeywordsABC transporter / ABCC4 / MRP4 / Topotecan bound / TRANSLOCASE
Function / homology
Function and homology information


guanine nucleotide transmembrane transporter activity / purine nucleotide transmembrane transporter activity / cAMP transport / 15-hydroxyprostaglandin dehydrogenase (NAD+) activity / urate transport / ABC-type bile acid transporter activity / platelet dense granule membrane / leukotriene transport / glutathione transmembrane transporter activity / prostaglandin transport ...guanine nucleotide transmembrane transporter activity / purine nucleotide transmembrane transporter activity / cAMP transport / 15-hydroxyprostaglandin dehydrogenase (NAD+) activity / urate transport / ABC-type bile acid transporter activity / platelet dense granule membrane / leukotriene transport / glutathione transmembrane transporter activity / prostaglandin transport / urate transmembrane transporter activity / ABC-type glutathione-S-conjugate transporter / ABC-type glutathione S-conjugate transporter activity / prostaglandin transmembrane transporter activity / platelet degranulation / ATPase-coupled inorganic anion transmembrane transporter activity / external side of apical plasma membrane / xenobiotic transmembrane transport / export across plasma membrane / ABC-type xenobiotic transporter / prostaglandin secretion / Paracetamol ADME / bile acid and bile salt transport / Translocases; Catalysing the translocation of other compounds; Linked to the hydrolysis of a nucleoside triphosphate / ABC-type xenobiotic transporter activity / Azathioprine ADME / xenobiotic transmembrane transporter activity / efflux transmembrane transporter activity / transport across blood-brain barrier / cilium assembly / ATPase-coupled transmembrane transporter activity / ABC-type transporter activity / xenobiotic metabolic process / ABC-family proteins mediated transport / transmembrane transport / Platelet degranulation / basolateral plasma membrane / apical plasma membrane / nucleolus / Golgi apparatus / ATP hydrolysis activity / ATP binding / membrane / plasma membrane
Similarity search - Function
ATP-binding cassette sub-family C member 4 / : / ABC transporter transmembrane region / ABC transporter type 1, transmembrane domain / ABC transporter integral membrane type-1 fused domain profile. / ABC transporter type 1, transmembrane domain superfamily / ABC transporter-like, conserved site / ABC transporters family signature. / ABC transporter / ABC transporter-like, ATP-binding domain ...ATP-binding cassette sub-family C member 4 / : / ABC transporter transmembrane region / ABC transporter type 1, transmembrane domain / ABC transporter integral membrane type-1 fused domain profile. / ABC transporter type 1, transmembrane domain superfamily / ABC transporter-like, conserved site / ABC transporters family signature. / ABC transporter / ABC transporter-like, ATP-binding domain / ATP-binding cassette, ABC transporter-type domain profile. / ATPases associated with a variety of cellular activities / AAA+ ATPase domain / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
ATP-binding cassette sub-family C member 4
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.9 Å
AuthorsRaj I / Bloch M / Pape TH / Taylor NMI
Funding support Denmark, 1 items
OrganizationGrant numberCountry
Other government Denmark
CitationJournal: Structure / Year: 2023
Title: Structural and mechanistic basis of substrate transport by the multidrug transporter MRP4.
Authors: Magnus Bloch / Isha Raj / Tillmann Pape / Nicholas M I Taylor /
Abstract: Multidrug resistance-associated protein 4 (MRP4) is an ATP-binding cassette (ABC) transporter expressed at multiple tissue barriers where it actively extrudes a wide variety of drug compounds. ...Multidrug resistance-associated protein 4 (MRP4) is an ATP-binding cassette (ABC) transporter expressed at multiple tissue barriers where it actively extrudes a wide variety of drug compounds. Overexpression of MRP4 provides resistance to clinically used antineoplastic agents, making it a highly attractive therapeutic target for countering multidrug resistance. Here, we report cryo-EM structures of multiple physiologically relevant states of lipid bilayer-embedded human MRP4, including complexes between MRP4 and two widely used chemotherapeutic agents and a complex between MRP4 and its native substrate. The structures display clear similarities and distinct differences in the coordination of these chemically diverse substrates and, in combination with functional and mutational analysis, reveal molecular details of the transport mechanism. Our study provides key insights into the unusually broad substrate specificity of MRP4 and constitutes an important contribution toward a general understanding of multidrug transporters.
History
DepositionDec 7, 2022-
Header (metadata) releaseAug 30, 2023-
Map releaseAug 30, 2023-
UpdateNov 15, 2023-
Current statusNov 15, 2023Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_16294.png

Downloads & links

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Map

FileDownload / File: emd_16294.map.gz / Format: CCP4 / Size: 216 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCryo-EM map for nanodisc-reconstituted wildtype human MRP4 in complex with TPT
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.83 Å/pix.
x 384 pix.
= 319.488 Å		0.83 Å/pix.
x 384 pix.
= 319.488 Å		0.83 Å/pix.
x 384 pix.
= 319.488 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.832 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.2
Minimum - Maximum-0.99712884 - 1.495419
Average (Standard dev.)-0.0003262941 (±0.030572409)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions384384384
Spacing384384384
CellA=B=C: 319.488 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_16294_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map 1 for nanodisc-reconstituted wildtype human MRP4...

Fileemd_16294_half_map_1.map
AnnotationHalf map 1 for nanodisc-reconstituted wildtype human MRP4 in complex with TPT
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map 2 for nanodisc-reconstituted wildtype human MRP4...

Fileemd_16294_half_map_2.map
AnnotationHalf map 2 for nanodisc-reconstituted wildtype human MRP4 in complex with TPT
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Cryo-EM structure of nanodisc-reconstituted wildtype human MRP4 (...

EntireName: Cryo-EM structure of nanodisc-reconstituted wildtype human MRP4 (in complex with topotecan)
Components
  • Organelle or cellular component: Cryo-EM structure of nanodisc-reconstituted wildtype human MRP4 (in complex with topotecan)
    • Protein or peptide: ATP-binding cassette sub-family C member 4
  • Ligand: (S)-10-[(DIMETHYLAMINO)METHYL]-4-ETHYL-4,9-DIHYDROXY-1H-PYRANO[3',4':6,7]INOLIZINO[1,2-B]-QUINOLINE-3,14(4H,12H)-DIONE

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Supramolecule #1: Cryo-EM structure of nanodisc-reconstituted wildtype human MRP4 (...

SupramoleculeName: Cryo-EM structure of nanodisc-reconstituted wildtype human MRP4 (in complex with topotecan)
type: organelle_or_cellular_component / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: ATP-binding cassette sub-family C member 4

MacromoleculeName: ATP-binding cassette sub-family C member 4 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
EC number: Translocases; Catalysing the translocation of other compounds; Linked to the hydrolysis of a nucleoside triphosphate
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 149.693922 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MLPVYQEVKP NPLQDANLCS RVFFWWLNPL FKIGHKRRLE EDDMYSVLPE DRSQHLGEEL QGFWDKEVLR AENDAQKPSL TRAIIKCYW KSYLVLGIFT LIEESAKVIQ PIFLGKIINY FENYDPMDSV ALNTAYAYAT VLTFCTLILA ILHHLYFYHV Q CAGMRLRV ...String:
MLPVYQEVKP NPLQDANLCS RVFFWWLNPL FKIGHKRRLE EDDMYSVLPE DRSQHLGEEL QGFWDKEVLR AENDAQKPSL TRAIIKCYW KSYLVLGIFT LIEESAKVIQ PIFLGKIINY FENYDPMDSV ALNTAYAYAT VLTFCTLILA ILHHLYFYHV Q CAGMRLRV AMCHMIYRKA LRLSNMAMGK TTTGQIVNLL SNDVNKFDQV TVFLHFLWAG PLQAIAVTAL LWMEIGISCL AG MAVLIIL LPLQSCFGKL FSSLRSKTAT FTDARIRTMN EVITGIRIIK MYAWEKSFSN LITNLRKKEI SKILRSSCLR GMN LASFFS ASKIIVFVTF TTYVLLGSVI TASRVFVAVT LYGAVRLTVT LFFPSAIERV SEAIVSIRRI QTFLLLDEIS QRNR QLPSD GKKMVHVQDF TAFWDKASET PTLQGLSFTV RPGELLAVVG PVGAGKSSLL SAVLGELAPS HGLVSVHGRI AYVSQ QPWV FSGTLRSNIL FGKKYEKERY EKVIKACALK KDLQLLEDGD LTVIGDRGTT LSGGQKARVN LARAVYQDAD IYLLDD PLS AVDAEVSRHL FELCICQILH EKITILVTHQ LQYLKAASQI LILKDGKMVQ KGTYTEFLKS GIDFGSLLKK DNEESEQ PP VPGTPTLRNR TFSESSVWSQ QSSRPSLKDG ALESQDTENV PVTLSEENRS EGKVGFQAYK NYFRAGAHWI VFIFLILL N TAAQVAYVLQ DWWLSYWANK QSMLNVTVNG GGNVTEKLDL NWYLGIYSGL TVATVLFGIA RSLLVFYVLV NSSQTLHNK MFESILKAPV LFFDRNPIGR ILNRFSKDIG HLDDLLPLTF LDFIQTLLQV VGVVSVAVAV IPWIAIPLVP LGIIFIFLRR YFLETSRDV KRLESTTRSP VFSHLSSSLQ GLWTIRAYKA EERCQELFDA HQDLHSEAWF LFLTTSRWFA VRLDAICAMF V IIVAFGSL ILAKTLDAGQ VGLALSYALT LMGMFQWCVR QSAEVENMMI SVERVIEYTD LEKEAPWEYQ KRPPPAWPHE GV IIFDNVN FMYSPGGPLV LKHLTALIKS QEKVGIVGRT GAGKSSLISA LFRLSEPEGK IWIDKILTTE IGLHDLRKKM SII PQEPVL FTGTMRKNLD PFNEHTDEEL WNALQEVQLK ETIEDLPGKM DTELAESGSN FSVGQRQLVC LARAILRKNQ ILII DEATA NVDPRTDELI QKKIREKFAH CTVLTIAHRL NTIIDSDKIM VLDSGRLKEY DEPYVLLQNK ESLFYKMVQQ LGKAE AAAL TETAKQVYFK RNYPHIGHTD HMVTNTSNGQ PSTLTIFETA L

UniProtKB: ATP-binding cassette sub-family C member 4

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Macromolecule #2: (S)-10-[(DIMETHYLAMINO)METHYL]-4-ETHYL-4,9-DIHYDROXY-1H-PYRANO[3'...

MacromoleculeName: (S)-10-[(DIMETHYLAMINO)METHYL]-4-ETHYL-4,9-DIHYDROXY-1H-PYRANO[3',4':6,7]INOLIZINO[1,2-B]-QUINOLINE-3,14(4H,12H)-DIONE
type: ligand / ID: 2 / Number of copies: 1 / Formula: TTC
Molecular weightTheoretical: 421.446 Da
Chemical component information

ChemComp-TTC:
(S)-10-[(DIMETHYLAMINO)METHYL]-4-ETHYL-4,9-DIHYDROXY-1H-PYRANO[3',4':6,7]INOLIZINO[1,2-B]-QUINOLINE-3,14(4H,12H)-DIONE / medication, inhibitor*YM / Topotecan

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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: 7.5
GridModel: Quantifoil R2/1 / Material: COPPER
VitrificationCryogen name: ETHANE / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV
DetailsThis sample was monodisperse

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 2.5 µm / Nominal defocus min: 0.5 µm / Nominal magnification: 96000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: FEI FALCON III (4k x 4k) / Detector mode: COUNTING / Number real images: 1449 / Average electron dose: 40.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 465447
Startup modelType of model: NONE
Initial angle assignmentType: OTHER / Software - Name: cryoSPARC (ver. 4.0.2)
Final angle assignmentType: OTHER / Software - Name: cryoSPARC (ver. 4.0.2)
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.9 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4.0.2) / Number images used: 56002
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementProtocol: AB INITIO MODEL
Output model

PDB-8bwq:
Cryo-EM structure of nanodisc-reconstituted wildtype human MRP4 (in complex with topotecan)

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