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- PDB-6fn1: Zosuquidar and UIC2 Fab complex of human-mouse chimeric ABCB1 (AB... -

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Basic information

Entry
Database: PDB / ID: 6fn1
TitleZosuquidar and UIC2 Fab complex of human-mouse chimeric ABCB1 (ABCB1HM)
Components
  • Human-mouse chimeric ABCB1 (ABCBHM)
  • UIC2 Antigen Binding Fragment Heavy Chain
  • UIC2 Antigen Binding Fragment Light chain
KeywordsMEMBRANE PROTEIN / Membrane Transport Protein ABCB1 ABC Exporter Small molecule inhibitor Antibody complex
Function / homology
Function and homology information


positive regulation of anion channel activity / ceramide translocation / terpenoid transport / ceramide floppase activity / carboxylic acid transmembrane transport / carboxylic acid transmembrane transporter activity / floppase activity / Abacavir transmembrane transport / external side of apical plasma membrane / regulation of response to osmotic stress ...positive regulation of anion channel activity / ceramide translocation / terpenoid transport / ceramide floppase activity / carboxylic acid transmembrane transport / carboxylic acid transmembrane transporter activity / floppase activity / Abacavir transmembrane transport / external side of apical plasma membrane / regulation of response to osmotic stress / Atorvastatin ADME / 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 / ABC-type xenobiotic transporter / transepithelial transport / P-type phospholipid transporter / ABC-type xenobiotic transporter activity / phospholipid translocation / Prednisone ADME / xenobiotic transmembrane transporter activity / efflux transmembrane transporter activity / transmembrane transporter activity / transport across blood-brain barrier / ATPase-coupled transmembrane transporter activity / xenobiotic metabolic process / regulation of chloride transport / stem cell proliferation / ABC-family proteins mediated transport / transmembrane transport / G2/M transition of mitotic cell cycle / response to xenobiotic stimulus / apical plasma membrane / ubiquitin protein ligase binding / cell surface / ATP hydrolysis activity / extracellular exosome / 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 / Immunoglobulins / Immunoglobulin-like / Sandwich / P-loop containing nucleoside triphosphate hydrolase / Mainly Beta
Similarity search - Domain/homology
Zosuquidar / ATP-dependent translocase ABCB1
Similarity search - Component
Biological speciesHomo sapiens (human)
Mus musculus (house mouse)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.58 Å
AuthorsAlam, A. / Locher, K.P.
Funding support Switzerland, United States, 3items
OrganizationGrant numberCountry
Swiss National Science Foundation Switzerland
European Molecular Biology Organization Switzerland
National Institutes of Health United States
CitationJournal: Proc Natl Acad Sci U S A / Year: 2018
Title: Structure of a zosuquidar and UIC2-bound human-mouse chimeric ABCB1.
Authors: Amer Alam / Raphael Küng / Julia Kowal / Robert A McLeod / Nina Tremp / Eugenia V Broude / Igor B Roninson / Henning Stahlberg / Kaspar P Locher /
Abstract: The multidrug transporter ABCB1 (P-glycoprotein) is an ATP-binding cassette transporter that has a key role in protecting tissues from toxic insult and contributes to multidrug extrusion from cancer ...The multidrug transporter ABCB1 (P-glycoprotein) is an ATP-binding cassette transporter that has a key role in protecting tissues from toxic insult and contributes to multidrug extrusion from cancer cells. Here, we report the near-atomic resolution cryo-EM structure of nucleotide-free ABCB1 trapped by an engineered disulfide cross-link between the nucleotide-binding domains (NBDs) and bound to the antigen-binding fragment of the human-specific inhibitory antibody UIC2 and to the third-generation ABCB1 inhibitor zosuquidar. Our structure reveals the transporter in an occluded conformation with a central, enclosed, inhibitor-binding pocket lined by residues from all transmembrane (TM) helices of ABCB1. The pocket spans almost the entire width of the lipid membrane and is occupied exclusively by two closely interacting zosuquidar molecules. The external, conformational epitope facilitating UIC2 binding is also visualized, providing a basis for its inhibition of substrate efflux. Additional cryo-EM structures suggest concerted movement of TM helices from both halves of the transporters associated with closing the NBD gap, as well as zosuquidar binding. Our results define distinct recognition interfaces of ABCB1 inhibitory agents, which may be exploited for therapeutic purposes.
History
DepositionFeb 2, 2018Deposition site: PDBE / Processing site: PDBE
Revision 1.0Feb 21, 2018Provider: repository / Type: Initial release
Revision 1.1Feb 28, 2018Group: Advisory / Author supporting evidence ...Advisory / Author supporting evidence / Database references / Derived calculations
Category: citation / pdbx_audit_support ...citation / pdbx_audit_support / pdbx_unobs_or_zero_occ_atoms / struct_conn
Item: _citation.journal_abbrev / _citation.pdbx_database_id_PubMed ..._citation.journal_abbrev / _citation.pdbx_database_id_PubMed / _citation.title / _pdbx_audit_support.funding_organization
Revision 1.2Mar 7, 2018Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last
Revision 1.3Dec 11, 2019Group: Other / Category: atom_sites
Item: _atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[2][2] / _atom_sites.fract_transf_matrix[3][3]
Revision 1.4Jul 29, 2020Group: Data collection / Derived calculations / Structure summary
Category: chem_comp / entity ...chem_comp / entity / pdbx_chem_comp_identifier / pdbx_entity_nonpoly / struct_conn / struct_site / struct_site_gen
Item: _chem_comp.name / _chem_comp.type ..._chem_comp.name / _chem_comp.type / _entity.pdbx_description / _pdbx_entity_nonpoly.name / _struct_conn.pdbx_role
Description: Carbohydrate remediation / Provider: repository / Type: Remediation

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Structure visualization

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Assembly

Deposited unit
A: Human-mouse chimeric ABCB1 (ABCBHM)
B: UIC2 Antigen Binding Fragment Light chain
C: UIC2 Antigen Binding Fragment Heavy Chain
hetero molecules


Theoretical massNumber of molelcules
Total (without water)188,1418
Polymers186,4223
Non-polymers1,7195
Water0
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: microscopy, gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area6960 Å2
ΔGint-32 kcal/mol
Surface area69250 Å2
MethodPISA

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Components

#1: Protein Human-mouse chimeric ABCB1 (ABCBHM)


Mass: 137719.953 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Cell line (production host): HEK / Production host: Homo sapiens (human) / References: UniProt: P08183*PLUS
#2: Antibody UIC2 Antigen Binding Fragment Light chain


Mass: 24321.039 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Cell line (production host): Hybridoma / Production host: Mus musculus (house mouse)
#3: Antibody UIC2 Antigen Binding Fragment Heavy Chain


Mass: 24381.281 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Cell line (production host): Hybridoma / Production host: Mus musculus (house mouse)
#4: Sugar ChemComp-NAG / 2-acetamido-2-deoxy-beta-D-glucopyranose / N-Acetylglucosamine


Type: D-saccharide, beta linking / Mass: 221.208 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Formula: C8H15NO6
IdentifierTypeProgram
DGlcpNAcbCONDENSED IUPAC CARBOHYDRATE SYMBOLGMML 1.0
N-acetyl-b-D-glucopyranosamineCOMMON NAMEGMML 1.0
b-D-GlcpNAcIUPAC CARBOHYDRATE SYMBOLPDB-CARE 1.0
GlcNAcSNFG CARBOHYDRATE SYMBOLGMML 1.0
#5: Chemical ChemComp-ZQU / Zosuquidar / Zosuquidar


Mass: 527.604 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C32H31F2N3O2 / Comment: antineoplastic*YM

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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

Component
IDNameTypeEntity IDParent-IDSource
1Human-mouse chimeric ABCB1 (ABCB1HM) complex with UIC2 fab and zosuquidarCOMPLEX#1-#30MULTIPLE SOURCES
2Human-mouse chimeric ABCB1 (ABCB1HM) complex with UIC2 fab and zosuquidarCOMPLEX#11RECOMBINANT
3Human-mouse chimeric ABCB1 (ABCB1HM) complex with UIC2 fab and zosuquidarCOMPLEX#2-#31RECOMBINANT
Molecular weightValue: 0.195 MDa / Experimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
12Homo sapiens (human)9606
23Mus musculus (house mouse)10090
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
12Homo sapiens (human)9606
23Mus musculus (house mouse)10090
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 FIELDBright-field microscopy
Image recordingElectron dose: 1.54 e/Å2 / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.12_2829: / Classification: refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.58 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 231969 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0113025
ELECTRON MICROSCOPYf_angle_d1.19217661
ELECTRON MICROSCOPYf_dihedral_angle_d7.7358441
ELECTRON MICROSCOPYf_chiral_restr0.0662016
ELECTRON MICROSCOPYf_plane_restr0.0082225

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