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- EMDB-19431: Cryo-em structure of the rat Multidrug resistance-associated prot... -

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Basic information

Entry
Database: EMDB / ID: EMD-19431
TitleCryo-em structure of the rat Multidrug resistance-associated protein 2 (rMrp2) in an autoinhibited state (nucleotide-free)
Map data
Sample
  • Organelle or cellular component: rat Multidrug resistance-associated protein 2 (rMrp2) in an autoinhibited state (nucleotide-free)
    • Protein or peptide: ATP-binding cassette sub-family C member 2
KeywordsMultidrug resistance-associated protein 2 (rMrp2) in an autoinhibited state (nucleotide-free) / TRANSPORT PROTEIN
Function / homology
Function and homology information


mercury ion transport / benzylpenicillin metabolic process / Aspirin ADME / Paracetamol ADME / Atorvastatin ADME / canalicular bile acid transport / intracellular canaliculus / antibiotic metabolic process / bilirubin transmembrane transporter activity / bilirubin transport ...mercury ion transport / benzylpenicillin metabolic process / Aspirin ADME / Paracetamol ADME / Atorvastatin ADME / canalicular bile acid transport / intracellular canaliculus / antibiotic metabolic process / bilirubin transmembrane transporter activity / bilirubin transport / xenobiotic export from cell / Heme degradation / response to antineoplastic agent / leukotriene transport / thyroid hormone transport / prostaglandin transport / detoxification of mercury ion / ABC-family proteins mediated transport / regulation of bile acid secretion / ABC-type glutathione-S-conjugate transporter / ABC-type glutathione S-conjugate transporter activity / intracellular chloride ion homeostasis / organic anion transport / xenobiotic transmembrane transport / organic anion transmembrane transporter activity / xenobiotic transport across blood-brain barrier / xenobiotic detoxification by transmembrane export across the plasma membrane / ABC-type xenobiotic transporter / intercellular canaliculus / transepithelial transport / response to arsenic-containing substance / bile acid and bile salt transport / Translocases; Catalysing the translocation of other compounds; Linked to the hydrolysis of a nucleoside triphosphate / cellular response to interleukin-6 / ABC-type xenobiotic transporter activity / bile acid signaling pathway / response to glucagon / response to steroid hormone / xenobiotic transmembrane transporter activity / ATPase-coupled transmembrane transporter activity / cellular response to interleukin-1 / xenobiotic catabolic process / cellular response to dexamethasone stimulus / female pregnancy / brush border membrane / transmembrane transport / response to organic cyclic compound / response to estrogen / cellular response to xenobiotic stimulus / response to estradiol / cellular response to tumor necrosis factor / response to oxidative stress / cellular response to lipopolysaccharide / response to lipopolysaccharide / response to xenobiotic stimulus / apical plasma membrane / protein domain specific binding / negative regulation of gene expression / cell surface / ATP hydrolysis activity / ATP binding / membrane / plasma membrane
Similarity search - Function
Multi drug resistance-associated protein / 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. ...Multi drug resistance-associated protein / 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 2
Similarity search - Component
Biological speciesRattus norvegicus (Norway rat)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.21 Å
AuthorsMazza T / Beis K
Funding support Canada, 1 items
OrganizationGrant numberCountry
Medical Research Council (MRC, Canada)MR/N020103/1 Canada
CitationJournal: Nat Commun / Year: 2024
Title: Structural basis for the modulation of MRP2 activity by phosphorylation and drugs.
Authors: Tiziano Mazza / Theodoros I Roumeliotis / Elena Garitta / David Drew / S Tamir Rashid / Cesare Indiveri / Jyoti S Choudhary / Kenneth J Linton / Konstantinos Beis /
Abstract: Multidrug resistance-associated protein 2 (MRP2/ABCC2) is a polyspecific efflux transporter of organic anions expressed in hepatocyte canalicular membranes. MRP2 dysfunction, in Dubin-Johnson ...Multidrug resistance-associated protein 2 (MRP2/ABCC2) is a polyspecific efflux transporter of organic anions expressed in hepatocyte canalicular membranes. MRP2 dysfunction, in Dubin-Johnson syndrome or by off-target inhibition, for example by the uricosuric drug probenecid, elevates circulating bilirubin glucuronide and is a cause of jaundice. Here, we determine the cryo-EM structure of rat Mrp2 (rMrp2) in an autoinhibited state and in complex with probenecid. The autoinhibited state exhibits an unusual conformation for this class of transporter in which the regulatory domain is folded within the transmembrane domain cavity. In vitro phosphorylation, mass spectrometry and transport assays show that phosphorylation of the regulatory domain relieves this autoinhibition and enhances rMrp2 transport activity. The in vitro data is confirmed in human hepatocyte-like cells, in which inhibition of endogenous kinases also reduces human MRP2 transport activity. The drug-bound state reveals two probenecid binding sites that suggest a dynamic interplay with autoinhibition. Mapping of the Dubin-Johnson mutations onto the rodent structure indicates that many may interfere with the transition between conformational states.
History
DepositionJan 17, 2024-
Header (metadata) releaseFeb 14, 2024-
Map releaseFeb 14, 2024-
UpdateMar 13, 2024-
Current statusMar 13, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_19431.png

Downloads & links

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Map

FileDownload / File: emd_19431.map.gz / Format: CCP4 / Size: 325 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 0.645 Å
Density
Contour LevelBy AUTHOR: 0.0486
Minimum - Maximum-0.5383817 - 0.9044224
Average (Standard dev.)0.0004114156 (±0.016901445)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderYXZ
Origin000
Dimensions440440440
Spacing440440440
CellA=B=C: 283.8 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_19431_half_map_1.map
Projections & Slices
AxesZYX

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Slices (1/2)
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Histogram (log scale)

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Half map: #1

Fileemd_19431_half_map_2.map
Projections & Slices
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Histogram

Histogram (log scale)

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Sample components

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Entire : rat Multidrug resistance-associated protein 2 (rMrp2) in an autoi...

EntireName: rat Multidrug resistance-associated protein 2 (rMrp2) in an autoinhibited state (nucleotide-free)
Components
  • Organelle or cellular component: rat Multidrug resistance-associated protein 2 (rMrp2) in an autoinhibited state (nucleotide-free)
    • Protein or peptide: ATP-binding cassette sub-family C member 2

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Supramolecule #1: rat Multidrug resistance-associated protein 2 (rMrp2) in an autoi...

SupramoleculeName: rat Multidrug resistance-associated protein 2 (rMrp2) in an autoinhibited state (nucleotide-free)
type: organelle_or_cellular_component / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Rattus norvegicus (Norway rat)

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Macromolecule #1: ATP-binding cassette sub-family C member 2

MacromoleculeName: ATP-binding cassette sub-family C member 2 / 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: Rattus norvegicus (Norway rat)
Molecular weightTheoretical: 173.571578 KDa
Recombinant expressionOrganism: Saccharomyces cerevisiae (brewer's yeast)
SequenceString: MDKFCNSTFW DLSLLESPEA DLPLCFEQTV LVWIPLGFLW LLAPWQLYSV YRSRTKRSSI TKFYLAKQVF VVFLLILAAI DLSLALTED TGQATVPPVR YTNPILYLCT WLLVLAVQHS RQWCVRKNSW FLSLFWILSV LCGVFQFQTL IRALLKDSKS N MAYSYLFF ...String:
MDKFCNSTFW DLSLLESPEA DLPLCFEQTV LVWIPLGFLW LLAPWQLYSV YRSRTKRSSI TKFYLAKQVF VVFLLILAAI DLSLALTED TGQATVPPVR YTNPILYLCT WLLVLAVQHS RQWCVRKNSW FLSLFWILSV LCGVFQFQTL IRALLKDSKS N MAYSYLFF VSYGFQIVLL ILTAFSGPSD STQTPSVTAS FLSSITFSWY DRTVLKGYKH PLTLEDVWDI DEGFKTRSVT SK FEAAMTK DLQKARQAFQ RRLQKSQRKP EATLHGLNKK QSQSQDVLVL EEAKKKSEKT TKDYPKSWLI KSLFKTFHVV ILK SFILKL IHDLLVFLNP QLLKLLIGFV KSSNSYVWFG YICAILMFAV TLIQSFCLQS YFQHCFVLGM CVRTTVMSSI YKKA LTLSN LARKQYTIGE TVNLMSVDSQ KLMDATNYMQ LVWSSVIQIT LSIFFLWREL GPSILAGVGV MVLLIPVNGV LATKI RNIQ VQNMKNKDKR LKIMNEILSG IKILKYFAWE PSFQEQVQGI RKKELKNLLR FGQLQSLLIF ILQITPILVS VVTFSV YVL VDSANVLNAE KAFTSITLFN ILRFPLSMLP MVTSSILQAS VSVDRLERYL GGDDLDTSAI RRVSNFDKAV KFSEASF TW DPDLEATIQD VNLDIKPGQL VAVVGTVGSG KSSLVSAMLG EMENVHGHIT IQGSTAYVPQ QSWIQNGTIK DNILFGSE Y NEKKYQQVLK ACALLPDLEI LPGGDMAEIG EKGINLSGGQ KQRVSLARAA YQDADIYILD DPLSAVDAHV GKHIFNKVV GPNGLLAGKT RIFVTHGIHF LPQVDEIVVL GKGTILEKGS YRDLLDKKGV FARNWKTFMK HSGPEGEATV NNDSEAEDDD DGLIPTMEE IPEDAASLAM RRENSLRRTL SRSSRSSSRR GKSLKNSLKI KNVNVLKEKE KEVEGQKLIK KEFVETGKVK F SIYLKYLQ AVGWWSILFI ILFYGLNNVA FIGSNLWLSA WTSDSDNLNG TNNSSSHRDM RIGVFGALGL AQGICLLIST LW SIYACRN ASKALHGQLL TNILRAPMRF FDTTPTGRIV NRFSGDISTV DDLLPQTLRS WMMCFFGIAG TLVMICMATP VFA IIIIPL SILYISVQVF YVATSRQLRR LDSVTKSPIY SHFSETVTGL PIIRAFEHQQ RFLAWNEKQI DINQKCVFSW ITSN RWLAI RLELVGNLVV FCSALLLVIY RKTLTGDVVG FVLSNALNIT QTLNWLVRMT SEAETNIVAV ERISEYINVE NEAPW VTDK RPPADWPRHG EIQFNNYQVR YRPELDLVLK GITCNIKSGE KVGVVGRTGA GKSSLTNCLF RILESAGGQI IIDGID VAS IGLHDLRERL TIIPQDPILF SGSLRMNLDP FNKYSDEEVW RALELAHLRS FVSGLQLGLL SEVTEGGDNL SIGQRQL LC LGRAVLRKSK ILVLDEATAA VDLETDSLIQ TTIRKEFSQC TVITIAHRLH TIMDSDKIMV LDNGKIVEYG SPEELLSN R GSFYLMAKEA GIENVNHTEL

UniProtKB: ATP-binding cassette sub-family C member 2

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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
VitrificationCryogen name: ETHANE

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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: 2.4 µm / Nominal defocus min: 1.2 µm
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 49.8 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: NONE
Initial angle assignmentType: ANGULAR RECONSTITUTION
Final angle assignmentType: ANGULAR RECONSTITUTION
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.21 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 282209

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Atomic model buiding 1

Initial modelPDB ID:

0-f1


Chain - Chain ID: A / Chain - Source name: AlphaFold / Chain - Initial model type: in silico model
RefinementProtocol: AB INITIO MODEL
Output model

PDB-8rq3:
Cryo-em structure of the rat Multidrug resistance-associated protein 2 (rMrp2) in an autoinhibited state (nucleotide-free)

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