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- PDB-8rq4: Cryo-em structure of the rat Multidrug resistance-associated prot... -

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Basic information

Entry
Database: PDB / ID: 8rq4
TitleCryo-em structure of the rat Multidrug resistance-associated protein 2 (rMrp2) in complex with probenecid
ComponentsATP-binding cassette sub-family C member 2
KeywordsTRANSPORT PROTEIN / Multidrug resistance-associated protein 2 (rMrp2) in an autoinhibited state (nucleotide-free)
Function / homology
Function and homology information


mercury ion transport / glucuronoside transport / benzylpenicillin metabolic process / Aspirin ADME / Paracetamol ADME / Atorvastatin ADME / response to cisplatin / proximal tubule development / antibiotic metabolic process / canalicular bile acid transport ...mercury ion transport / glucuronoside transport / benzylpenicillin metabolic process / Aspirin ADME / Paracetamol ADME / Atorvastatin ADME / response to cisplatin / proximal tubule development / antibiotic metabolic process / canalicular bile acid transport / intracellular canaliculus / pigment metabolic process / response to wortmannin / glutathione transport / bilirubin transmembrane transporter activity / Heme degradation / xenobiotic export from cell / : / response to antineoplastic agent / ABC-family protein mediated transport / mRNA metabolic process / detoxification of mercury ion / intracellular chloride ion homeostasis / response to peptide / thyroid hormone transport / leukotriene transport / toxin transmembrane transporter activity / prostaglandin transport / response to mercury ion / ABC-type glutathione-S-conjugate transporter / carboxylic acid transmembrane transporter activity / ABC-type glutathione S-conjugate transporter activity / bile acid metabolic process / cardiac muscle cell differentiation / xenobiotic transport across blood-brain barrier / metal ion transport / bilirubin transport / intercellular canaliculus / xenobiotic detoxification by transmembrane export across the plasma membrane / response to arsenic-containing substance / xenobiotic transmembrane transport / : / heme catabolic process / transepithelial transport / cellular response to interleukin-6 / response to glucagon / response to steroid hormone / response to growth hormone / ABC-type xenobiotic transporter / Translocases; Catalysing the translocation of other compounds; Linked to the hydrolysis of a nucleoside triphosphate / ABC-type xenobiotic transporter activity / bile acid and bile salt transport / cellular response to dexamethasone stimulus / xenobiotic transmembrane transporter activity / cellular response to interleukin-1 / ATPase-coupled transmembrane transporter activity / xenobiotic catabolic process / xenobiotic transport / glutathione metabolic process / phosphatidylinositol 3-kinase/protein kinase B signal transduction / cellular response to xenobiotic stimulus / brush border membrane / female pregnancy / cellular response to tumor necrosis factor / response to insulin / response to estrogen / response to toxic substance / gene expression / transmembrane transport / heart development / response to estradiol / cellular response to lipopolysaccharide / response to lipopolysaccharide / response to oxidative stress / vesicle / apical plasma membrane / response to xenobiotic stimulus / negative regulation of gene expression / protein domain specific binding / cell surface / ATP hydrolysis activity / ATP binding / membrane / plasma membrane
Similarity search - Function
Multi drug resistance-associated protein / : / ABC transporter TMD0 domain / : / 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. ...Multi drug resistance-associated protein / : / ABC transporter TMD0 domain / : / 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
4-(dipropylsulfamoyl)benzoic acid / CHOLESTEROL HEMISUCCINATE / ATP-binding cassette sub-family C member 2
Similarity search - Component
Biological speciesRattus norvegicus (Norway rat)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.45 Å
AuthorsMazza, T. / Beis, K.
Funding support Canada, 1items
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, 2024Deposition site: PDBE / Processing site: PDBE
Revision 1.0Feb 14, 2024Provider: repository / Type: Initial release
Revision 1.0Feb 14, 2024Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
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Revision 1.1Mar 13, 2024Group: Database references / Category: citation / citation_author
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Revision 1.2Nov 6, 2024Group: Data collection / Structure summary
Category: em_admin / pdbx_entry_details / pdbx_modification_feature
Item: _em_admin.last_update / _pdbx_entry_details.has_protein_modification
Revision 1.3Jul 2, 2025Group: Data collection / Category: em_admin / em_software / Item: _em_admin.last_update / _em_software.name
Revision 1.1Jul 2, 2025Data content type: EM metadata / Data content type: EM metadata / EM metadata / Group: Data processing / Experimental summary / Data content type: EM metadata / EM metadata / Category: em_admin / em_software / Data content type: EM metadata / EM metadata / Item: _em_admin.last_update / _em_software.name

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: ATP-binding cassette sub-family C member 2
hetero molecules


Theoretical massNumber of molelcules
Total (without water)174,6294
Polymers173,5721
Non-polymers1,0573
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein ATP-binding cassette sub-family C member 2 / Canalicular multidrug resistance protein / Canalicular multispecific organic anion transporter 1 / ...Canalicular multidrug resistance protein / Canalicular multispecific organic anion transporter 1 / Multidrug resistance-associated protein 2


Mass: 173571.578 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Rattus norvegicus (Norway rat) / Gene: Abcc2, Cmoat, Cmrp, Mrp2 / Production host: Saccharomyces cerevisiae (brewer's yeast)
References: UniProt: Q63120, Translocases; Catalysing the translocation of other compounds; Linked to the hydrolysis of a nucleoside triphosphate, ABC-type xenobiotic transporter, ABC-type ...References: UniProt: Q63120, Translocases; Catalysing the translocation of other compounds; Linked to the hydrolysis of a nucleoside triphosphate, ABC-type xenobiotic transporter, ABC-type glutathione-S-conjugate transporter
#2: Chemical ChemComp-Y01 / CHOLESTEROL HEMISUCCINATE


Mass: 486.726 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C31H50O4 / Feature type: SUBJECT OF INVESTIGATION
#3: Chemical ChemComp-RTO / 4-(dipropylsulfamoyl)benzoic acid


Mass: 285.359 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C13H19NO4S / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY
Has protein modificationY

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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: Cryo-em structure of the rat Multidrug resistance-associated protein 2 (rMrp2) in complex with probenecid
Type: ORGANELLE OR CELLULAR COMPONENT / Entity ID: #1 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Rattus norvegicus (Norway rat)
Source (recombinant)Organism: Saccharomyces cerevisiae (brewer's yeast)
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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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 defocus max: 2400 nm / Nominal defocus min: 1200 nm
Image recordingElectron dose: 52.8 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

EM softwareName: PHENIX / Category: model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.45 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 247763 / Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL
Atomic model buildingPDB-ID: 8RQ3

8rq3


Pdb chain-ID: A / Accession code: 8RQ3 / Source name: PDB / Type: experimental model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00611449
ELECTRON MICROSCOPYf_angle_d0.6415538
ELECTRON MICROSCOPYf_dihedral_angle_d4.7941531
ELECTRON MICROSCOPYf_chiral_restr0.0431816
ELECTRON MICROSCOPYf_plane_restr0.0041916

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