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- PDB-6fn4: Apo form of UIC2 Fab complex of human-mouse chimeric ABCB1 (ABCB1HM) -

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Basic information

Entry
Database: PDB / ID: 6fn4
TitleApo form of UIC2 Fab complex of human-mouse chimeric ABCB1 (ABCB1HM)
Components
  • Apo form of Human-mouse chimeric ABCB1 (ABCB1HM) in complex with Antigen binding fragment of UIC2.
  • UIC2 Antigen Binding Fragment Light chain
  • UIC2 Antigen binding fragment Heavy chain
KeywordsMEMBRANE PROTEIN / Membrane Transport Protein ABCB1 ABC Exporter Small molecule inhibitor Antibody complex
Function / homology
Function and homology information


Abacavir transmembrane transport / ABC-family proteins mediated transport / ceramide translocation / phosphatidylethanolamine flippase activity / ceramide floppase activity / positive regulation of anion channel activity / phosphatidylcholine floppase activity / ABC-type xenobiotic transporter / P-type phospholipid transporter / regulation of response to osmotic stress ...Abacavir transmembrane transport / ABC-family proteins mediated transport / ceramide translocation / phosphatidylethanolamine flippase activity / ceramide floppase activity / positive regulation of anion channel activity / phosphatidylcholine floppase activity / ABC-type xenobiotic transporter / P-type phospholipid transporter / regulation of response to osmotic stress / phospholipid translocation / stem cell proliferation / ATPase-coupled transmembrane transporter activity / transporter activity / regulation of chloride transport / transmembrane transport / apical plasma membrane / G2/M transition of mitotic cell cycle / ATPase activity / response to drug / cell surface / extracellular exosome / membrane / integral component of membrane / ATP binding / plasma membrane
ABC transporter type 1, transmembrane domain superfamily / Type I protein exporter / AAA+ ATPase domain / ABC transporter type 1, transmembrane domain / ABC transporter, conserved site / P-loop containing nucleoside triphosphate hydrolase / Multidrug resistance protein 1 / ABC transporter / ABC transporter transmembrane region / ABC transporters family signature. ...ABC transporter type 1, transmembrane domain superfamily / Type I protein exporter / AAA+ ATPase domain / ABC transporter type 1, transmembrane domain / ABC transporter, conserved site / P-loop containing nucleoside triphosphate hydrolase / Multidrug resistance protein 1 / ABC transporter / ABC transporter transmembrane region / ABC transporters family signature. / ATP-binding cassette, ABC transporter-type domain profile. / ABC transporter integral membrane type-1 fused domain profile. / ABC transporter-like
ATP-dependent translocase ABCB1
Biological speciesHomo sapiens (human)
Mus musculus (house mouse)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.14 Å
AuthorsAlam, A. / Locher, K.P.
Funding supportSwitzerland , United States , 3件
OrganizationGrant numberCountry
Swiss National Science FoundationSwitzerland
European Molecular Biology OrganizationSwitzerland
National Institutes of HealthUnited 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.
Validation Report
SummaryFull reportAbout validation report
DateDeposition: Feb 2, 2018 / Release: Feb 21, 2018
RevisionDateData content typeGroupCategoryItemProviderType
1.0Feb 21, 2018Structure modelrepositoryInitial release
1.1Feb 28, 2018Structure modelAuthor supporting evidence / Database referencescitation / pdbx_audit_support_citation.journal_abbrev / _citation.pdbx_database_id_PubMed / _citation.title / _pdbx_audit_support.funding_organization
1.2Mar 7, 2018Structure modelDatabase referencescitation_citation.journal_volume / _citation.page_first / _citation.page_last

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Assembly

Deposited unit
A: Apo form of Human-mouse chimeric ABCB1 (ABCB1HM) in complex with Antigen binding fragment of UIC2.
B: UIC2 Antigen Binding Fragment Light chain
C: UIC2 Antigen binding fragment Heavy chain
hetero molecules


Theoretical massNumber of molelcules
Total (without water)191,3977
Polymers189,9853
Non-polymers1,4124
Water0
1


TypeNameSymmetry operationNumber
identity operation1_5551
Buried area7940 Å2
ΔGint-37 kcal/mol
Surface area75380 Å2
MethodPISA

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Components

#1: Protein/peptide Apo form of Human-mouse chimeric ABCB1 (ABCB1HM) in complex with Antigen binding fragment of UIC2.


Mass: 141283.156 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Production host: Homo sapiens (human) / References: UniProt: P08183*PLUS
#2: Protein/peptide 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) / Production host: Mus musculus (house mouse)
#3: Protein/peptide 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) / Production host: Mus musculus (house mouse)
#4: Chemical ChemComp-NAG / N-ACETYL-D-GLUCOSAMINE


Mass: 221.208 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Formula: C8H15NO6 / N-Acetylglucosamine
#5: Chemical ChemComp-3PE / 1,2-DIACYL-SN-GLYCERO-3-PHOSPHOETHANOLAMINE / 3-SN-PHOSPHATIDYLETHANOLAMINE


Mass: 748.065 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C41H82NO8P / Comment: phospholipid *YM
Sequence detailsUniprot IDs: Human ABCB1 P08183. Mouse ABCB1 P21447

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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

Name: Apo Human-mouse chimeric ABCB1 (ABCB1HM) complex with UIC2 fab / Type: COMPLEX

IDEntity IDParent-IDSource
11, 2, 30MULTIPLE SOURCES
211RECOMBINANT
32, 31RECOMBINANT
Molecular weight

Experimental value: NO

IDEntity assembly-IDValue (°)
110.195 MDa
220.142 MDa
330.0487 MDa
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: 0.9 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: 4.14 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 517053 / Symmetry type: POINT
Refine LS restraints

Refinement-ID: ELECTRON MICROSCOPY

TypeDev idealNumber
f_bond_d0.01212971
f_angle_d0.72217559
f_dihedral_angle_d6.8437672
f_chiral_restr0.0462007
f_plane_restr0.0052218

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