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- PDB-8pee: ABCB1 L335C mutant (mABCB1) in the inward facing state bound to AAC -

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Basic information

Entry
Database: PDB / ID: 8pee
TitleABCB1 L335C mutant (mABCB1) in the inward facing state bound to AAC
ComponentsATP-dependent translocase ABCB1
KeywordsMEMBRANE PROTEIN / ABC transporter
Function / homology
Function and homology information


hormone transport / cellular response to borneol / response to codeine / response to cyclosporin A / Atorvastatin ADME / cellular response to mycotoxin / daunorubicin transport / positive regulation of response to drug / response to quercetin / cellular response to external biotic stimulus ...hormone transport / cellular response to borneol / response to codeine / response to cyclosporin A / Atorvastatin ADME / cellular response to mycotoxin / daunorubicin transport / positive regulation of response to drug / response to quercetin / cellular response to external biotic stimulus / regulation of intestinal absorption / response to antineoplastic agent / Prednisone ADME / positive regulation of establishment of Sertoli cell barrier / ceramide translocation / terpenoid transport / ceramide floppase activity / response to glycoside / carboxylic acid transmembrane transport / carboxylic acid transmembrane transporter activity / floppase activity / establishment of blood-retinal barrier / protein localization to bicellular tight junction / regulation of response to osmotic stress / cellular response to L-glutamate / ABC-family proteins mediated transport / establishment of blood-brain barrier / response to thyroxine / phosphatidylcholine floppase activity / phosphatidylethanolamine flippase activity / xenobiotic transport across blood-brain barrier / xenobiotic detoxification by transmembrane export across the plasma membrane / export across plasma membrane / response to vitamin D / ABC-type xenobiotic transporter / intercellular canaliculus / transepithelial transport / P-type phospholipid transporter / ABC-type xenobiotic transporter activity / response to vitamin A / response to glucagon / intestinal absorption / phospholipid translocation / cellular response to antibiotic / maintenance of blood-brain barrier / cellular hyperosmotic salinity response / cellular response to alkaloid / xenobiotic transmembrane transporter activity / efflux transmembrane transporter activity / transmembrane transporter activity / ATPase-coupled transmembrane transporter activity / response to cadmium ion / lactation / cellular response to dexamethasone stimulus / placenta development / regulation of chloride transport / stem cell proliferation / cellular response to estradiol stimulus / brush border membrane / circadian rhythm / G2/M transition of mitotic cell cycle / cellular response to tumor necrosis factor / cellular response to lipopolysaccharide / response to hypoxia / response to xenobiotic stimulus / apical plasma membrane / ubiquitin protein ligase binding / cell surface / ATP hydrolysis activity / ATP binding / membrane / plasma membrane / cytoplasm
Similarity search - Function
Type 1 protein exporter / 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. ...Type 1 protein exporter / 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
Chem-JIZ / Chem-JJI / CHOLESTEROL HEMISUCCINATE / ATP-dependent translocase ABCB1
Similarity search - Component
Biological speciesMus musculus (house mouse)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.8 Å
AuthorsParey, K. / Januliene, D. / Gewering, T. / Moeller, A.
Funding support Germany, 2items
OrganizationGrant numberCountry
German Research Foundation (DFG)CRC944 Germany
German Research Foundation (DFG)INST 190-196-1 FUGG Germany
CitationJournal: Elife / Year: 2024
Title: Tracing the substrate translocation mechanism in P-glycoprotein.
Authors: Theresa Gewering / Deepali Waghray / Kristian Parey / Hendrik Jung / Nghi N B Tran / Joel Zapata / Pengyi Zhao / Hao Chen / Dovile Januliene / Gerhard Hummer / Ina Urbatsch / Arne Moeller / Qinghai Zhang /
Abstract: P-glycoprotein (Pgp) is a prototypical ATP-binding cassette (ABC) transporter of great biological and clinical significance.Pgp confers cancer multidrug resistance and mediates the bioavailability ...P-glycoprotein (Pgp) is a prototypical ATP-binding cassette (ABC) transporter of great biological and clinical significance.Pgp confers cancer multidrug resistance and mediates the bioavailability and pharmacokinetics of many drugs (Juliano and Ling, 1976; Ueda et al., 1986; Sharom, 2011). Decades of structural and biochemical studies have provided insights into how Pgp binds diverse compounds (Loo and Clarke, 2000; Loo et al., 2009; Aller et al., 2009; Alam et al., 2019; Nosol et al., 2020; Chufan et al., 2015), but how they are translocated through the membrane has remained elusive. Here, we covalently attached a cyclic substrate to discrete sites of Pgp and determined multiple complex structures in inward- and outward-facing states by cryoEM. In conjunction with molecular dynamics simulations, our structures trace the substrate passage across the membrane and identify conformational changes in transmembrane helix 1 (TM1) as regulators of substrate transport. In mid-transport conformations, TM1 breaks at glycine 72. Mutation of this residue significantly impairs drug transport of Pgp in vivo, corroborating the importance of its regulatory role. Importantly, our data suggest that the cyclic substrate can exit Pgp without the requirement of a wide-open outward-facing conformation, diverting from the common efflux model for Pgp and other ABC exporters. The substrate transport mechanism of Pgp revealed here pinpoints critical targets for future drug discovery studies of this medically relevant system.
History
DepositionJun 13, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0Mar 20, 2024Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: ATP-dependent translocase ABCB1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)149,6858
Polymers146,0641
Non-polymers3,6217
Water0
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area0 Å2
ΔGint0 kcal/mol
Surface area55610 Å2
MethodPISA

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Components

#1: Protein ATP-dependent translocase ABCB1 / ATP-binding cassette sub-family B member 1A / MDR1A / Multidrug resistance protein 1A / Multidrug ...ATP-binding cassette sub-family B member 1A / MDR1A / Multidrug resistance protein 1A / Multidrug resistance protein 3 / P-glycoprotein 3 / Phospholipid transporter ABCB1


Mass: 146063.859 Da / Num. of mol.: 1
Mutation: GLN A 83 UNP P21447 ASN 83 ENGINEERED MUTATION GLN A 87 UNP P21447 ASN 87 ENGINEERED MUTATION GLN A 90 UNP P21447 ASN 90 ENGINEERED MUTATION ALA A 133 UNP P21447 CYS 133 ENGINEERED MUTATION ...Mutation: GLN A 83 UNP P21447 ASN 83 ENGINEERED MUTATION GLN A 87 UNP P21447 ASN 87 ENGINEERED MUTATION GLN A 90 UNP P21447 ASN 90 ENGINEERED MUTATION ALA A 133 UNP P21447 CYS 133 ENGINEERED MUTATION CYS A 335 UNP P21447 LEU 335 ENGINEERED MUTATION GLY A 427 UNP P21447 CYS 427 ENGINEERED MUTATION ALA A 713 UNP P21447 CYS 713 ENGINEERED MUTATION ALA A 952 UNP P21447 CYS 952 ENGINEERED MUTATION GLY A 1070 UNP P21447 CYS 1070 ENGINEERED MUTATION ARG A 1121 UNP P21447 CYS 1121 ENGINEERED MUTATION VAL A 1223 UNP P21447 CYS 1223 ENGINEERED MUTATION
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Gene: Abcb1a, Abcb4, Mdr1a, Mdr3, Pgy-3, Pgy3 / Production host: Komagataella pastoris (fungus)
References: UniProt: P21447, ABC-type xenobiotic transporter, P-type phospholipid transporter
#2: Chemical ChemComp-JJI / (4S,11S,18S)-4-[[(2,4-dinitrophenyl)disulfanyl]methyl]-11,18-dimethyl-6,13,20-trithia-3,10,17,22,23,24-hexazatetracyclo[17.2.1.1^{5,8}.1^{12,15}]tetracosa-1(21),5(24),7,12(23),14,19(22)-hexaene-2,9,16-trione


Type: peptide-like / Mass: 692.790 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C24H20N8O7S5 / Feature type: SUBJECT OF INVESTIGATION
#3: Chemical ChemComp-JIZ / (4~{S},11~{S},18~{S})-4,11-dimethyl-18-(sulfanylmethyl)-6,13,20-trithia-3,10,17,22,23,24-hexazatetracyclo[17.2.1.1^{5,8}.1^{12,15}]tetracosa-1(21),5(24),7,12(23),14,19(22)-hexaene-2,9,16-trione


Mass: 494.634 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C18H18N6O3S4 / Feature type: SUBJECT OF INVESTIGATION
#4: Chemical
ChemComp-Y01 / CHOLESTEROL HEMISUCCINATE


Mass: 486.726 Da / Num. of mol.: 5 / Source method: obtained synthetically / Formula: C31H50O4
Has ligand of interestY

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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: ABCB1P-glycoprotein / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 140 kDa/nm / Experimental value: NO
Source (natural)Organism: Mus musculus (house mouse)
Source (recombinant)Organism: Komagataella pastoris (fungus)
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: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 2500 nm / Nominal defocus min: 1000 nm
Image recordingElectron dose: 75 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

EM software
IDNameVersionCategory
7Coot0.9.3model fitting
9PHENIX1.19.2model refinement
13cryoSPARC3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.8 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 218872 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL
Atomic model buildingPDB-ID: 6C0V

6c0v


Accession code: 6C0V / Source name: PDB / Type: experimental model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0039207
ELECTRON MICROSCOPYf_angle_d0.58812456
ELECTRON MICROSCOPYf_dihedral_angle_d6.051279
ELECTRON MICROSCOPYf_chiral_restr0.0411436
ELECTRON MICROSCOPYf_plane_restr0.0031575

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