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- PDB-8bjf: Cryo-EM structure of nanodisc-reconstituted wildtype human MRP4 (... -

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Basic information

Entry
Database: PDB / ID: 8bjf
TitleCryo-EM structure of nanodisc-reconstituted wildtype human MRP4 (inward-facing conformation)
ComponentsATP-binding cassette sub-family C member 4
KeywordsTRANSLOCASE / ABC transporter / ABCC4 / MRP4
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
CHOLESTEROL / CHOLESTEROL HEMISUCCINATE / ATP-binding cassette sub-family C member 4
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3 Å
AuthorsRaj, I. / Bloch, M. / Pape, T.H. / Taylor, N.M.I.
Funding support Denmark, 1items
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
DepositionNov 4, 2022Deposition site: PDBE / Processing site: PDBE
Revision 1.0Aug 30, 2023Provider: repository / Type: Initial release
Revision 1.1Sep 20, 2023Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.identifier_ORCID / _citation_author.name
Revision 1.2Nov 15, 2023Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last

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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 4
hetero molecules


Theoretical massNumber of molelcules
Total (without water)152,4147
Polymers149,6941
Non-polymers2,7206
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area1360 Å2
ΔGint1 kcal/mol
Surface area53290 Å2

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Components

#1: Protein ATP-binding cassette sub-family C member 4 / MRP/cMOAT-related ABC transporter / Multi-specific organic anion transporter B / MOAT-B / Multidrug ...MRP/cMOAT-related ABC transporter / Multi-specific organic anion transporter B / MOAT-B / Multidrug resistance-associated protein 4


Mass: 149693.922 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: Polypeptide Chain / Source: (gene. exp.) Homo sapiens (human) / Gene: ABCC4, MOATB, MRP4 / Cell line (production host): HEK / Production host: Homo sapiens (human)
References: UniProt: O15439, Translocases; Catalysing the translocation of other compounds; Linked to the hydrolysis of a nucleoside triphosphate, ABC-type xenobiotic transporter, ABC-type ...References: UniProt: O15439, 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-CLR / CHOLESTEROL / Cholesterol


Mass: 386.654 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C27H46O
#3: Chemical
ChemComp-Y01 / CHOLESTEROL HEMISUCCINATE


Mass: 486.726 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C31H50O4
Has ligand of interestN

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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 nanodisc-reconstituted wildtype human MRP4 (inward-facing conformation)
Type: ORGANELLE OR CELLULAR COMPONENT / Entity ID: #1 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Homo sapiens (human) / Cell: HEK / Plasmid: BacMam
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: This sample was monodisperse
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R2/1
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Chamber temperature: 277 K

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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 FIELDBright-field microscopy / Nominal magnification: 96000 X / Nominal defocus max: 2500 nm / Nominal defocus min: 500 nm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 40 e/Å2 / Detector mode: COUNTING / Film or detector model: FEI FALCON III (4k x 4k) / Num. of real images: 5391

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Processing

Software
NameVersionClassificationNB
phenix.real_space_refine1.20.1_4487refinement
PHENIX1.20.1_4487refinement
EM software
IDNameVersionCategory
1cryoSPARC4.0.2particle selection
2EPUimage acquisition
4cryoSPARC4.0.2CTF correction
7UCSF ChimeraX1.3model fitting
8Coot0.9.6 ELmodel fitting
9ISOLDEmodel fitting
11PHENIX1.20.1model refinement
12cryoSPARC4.0.2initial Euler assignment
13cryoSPARC4.0.2final Euler assignment
15cryoSPARC4.0.23D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 4200342
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 725758 / Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 81.89 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0039965
ELECTRON MICROSCOPYf_angle_d0.6113541
ELECTRON MICROSCOPYf_dihedral_angle_d13.6113622
ELECTRON MICROSCOPYf_chiral_restr0.0431585
ELECTRON MICROSCOPYf_plane_restr0.0081656

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