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- PDB-8p7w: Structure of 5D3-Fab and nanobody(Nb8)-bound ABCG2 -

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Basic information

Entry
Database: PDB / ID: 8p7w
TitleStructure of 5D3-Fab and nanobody(Nb8)-bound ABCG2
Components
  • 5D3(Fab) heavy chain variable domain
  • 5D3(Fab) light chain variable domain
  • ATP-binding cassette sub-family G member 2
  • NanobodySingle-domain antibody
KeywordsMEMBRANE PROTEIN
Function / homology
Function and homology information


biotin transmembrane transporter activity / biotin transport / riboflavin transport / riboflavin transmembrane transporter activity / renal urate salt excretion / urate metabolic process / urate transmembrane transporter activity / Abacavir transmembrane transport / organic anion transport / external side of apical plasma membrane ...biotin transmembrane transporter activity / biotin transport / riboflavin transport / riboflavin transmembrane transporter activity / renal urate salt excretion / urate metabolic process / urate transmembrane transporter activity / Abacavir transmembrane transport / organic anion transport / external side of apical plasma membrane / organic anion transmembrane transporter activity / xenobiotic transport across blood-brain barrier / export across plasma membrane / ABC-type xenobiotic transporter / Paracetamol ADME / Ciprofloxacin ADME / transepithelial transport / cellular detoxification / ABC-type xenobiotic transporter activity / NFE2L2 regulating MDR associated enzymes / Heme biosynthesis / Heme degradation / lipid transport / xenobiotic transmembrane transporter activity / efflux transmembrane transporter activity / transport across blood-brain barrier / ATPase-coupled transmembrane transporter activity / mitochondrial membrane / brush border membrane / Iron uptake and transport / transmembrane transport / membrane raft / apical plasma membrane / ATP hydrolysis activity / protein homodimerization activity / nucleoplasm / ATP binding / identical protein binding / plasma membrane
Similarity search - Function
ABC transporter family G domain / ABC-2 type transporter / ABC-2 type transporter / ABC-2 type transporter / 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
Broad substrate specificity ATP-binding cassette transporter ABCG2
Similarity search - Component
Biological speciesHomo sapiens (human)
Lama glama (llama)
Mus musculus (house mouse)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.04 Å
AuthorsIrobalieva, R.N. / Manolaridis, I. / Jackson, S.M. / Ni, D. / Pardon, E. / Stahlberg, H. / Steyaert, J. / Locher, K.P.
Funding support Switzerland, 1items
OrganizationGrant numberCountry
Swiss National Science Foundation Switzerland
CitationJournal: J Mol Biol / Year: 2023
Title: Structural Basis of the Allosteric Inhibition of Human ABCG2 by Nanobodies.
Authors: Rossitza N Irobalieva / Ioannis Manolaridis / Scott M Jackson / Dongchun Ni / Els Pardon / Henning Stahlberg / Jan Steyaert / Kaspar P Locher /
Abstract: ABCG2 is an ATP-binding cassette transporter that exports a wide range of xenobiotic compounds and has been recognized as a contributing factor for multidrug resistance in cancer cells. Substrate and ...ABCG2 is an ATP-binding cassette transporter that exports a wide range of xenobiotic compounds and has been recognized as a contributing factor for multidrug resistance in cancer cells. Substrate and inhibitor interactions with ABCG2 have been extensively studied and small molecule inhibitors have been developed that prevent the export of anticancer drugs from tumor cells. Here, we explore the potential for inhibitors that target sites other than the substrate binding pocket of ABCG2. We developed novel nanobodies against ABCG2 and used functional analyses to select three inhibitory nanobodies (Nb8, Nb17 and Nb96) for structural studies by single particle cryo-electron microscopy. Our results showed that these nanobodies allosterically bind to different regions of the nucleotide binding domains. Two copies of Nb8 bind to the apex of the NBDs preventing them from fully closing. Nb17 binds near the two-fold axis of the transporter and interacts with both NBDs. Nb96 binds to the side of the NBD and immobilizes a region connected to key motifs involved in ATP binding and hydrolysis. All three nanobodies prevent the transporter from undergoing conformational changes required for substrate transport. These findings advance our understanding of the molecular basis of modulation of ABCG2 by external binders, which may contribute to the development of a new generation of inhibitors. Furthermore, this is the first example of modulation of human multidrug resistance transporters by nanobodies.
History
DepositionMay 31, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0Aug 30, 2023Provider: repository / Type: Initial release
Revision 1.1Sep 13, 2023Group: Database references / Category: citation / Item: _citation.journal_volume / _citation.title

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: ATP-binding cassette sub-family G member 2
B: ATP-binding cassette sub-family G member 2
C: Nanobody
D: 5D3(Fab) heavy chain variable domain
E: 5D3(Fab) light chain variable domain
F: 5D3(Fab) heavy chain variable domain
G: 5D3(Fab) light chain variable domain
Y: Nanobody


Theoretical massNumber of molelcules
Total (without water)266,6118
Polymers266,6118
Non-polymers00
Water0
1


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

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Components

#1: Protein ATP-binding cassette sub-family G member 2 / Breast cancer resistance protein / CDw338 / Mitoxantrone resistance-associated protein / Placenta- ...Breast cancer resistance protein / CDw338 / Mitoxantrone resistance-associated protein / Placenta-specific ATP-binding cassette transporter / Urate exporter


Mass: 72385.852 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: ABCG2, ABCP, BCRP, BCRP1, MXR / Production host: Homo sapiens (human)
References: UniProt: Q9UNQ0, ABC-type xenobiotic transporter
#2: Antibody Nanobody / Single-domain antibody


Mass: 13481.859 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Lama glama (llama) / Production host: Escherichia coli (E. coli)
#3: Antibody 5D3(Fab) heavy chain variable domain


Mass: 23843.633 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Production host: Mus musculus (house mouse)
#4: Antibody 5D3(Fab) light chain variable domain


Mass: 23594.016 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Production host: Mus musculus (house mouse)

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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
1Structure of nanobody (Nb8)-bound ABCG2COMPLEXall0MULTIPLE SOURCES
2ATP-binding cassette sub-family G member 2COMPLEX#11RECOMBINANT
3NanobodySingle-domain antibodyCOMPLEX#21RECOMBINANT
45D3(Fab) heavy and light chain variable domainCOMPLEX#3-#41RECOMBINANT
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
22Homo sapiens (human)9606
33Lama glama (llama)9844
44Mus musculus (house mouse)10090
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
22Homo sapiens (human)9606
33Escherichia coli (E. coli)562
44Mus musculus (house mouse)10090
Buffer solutionpH: 7.5
SpecimenConc.: 1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationCryogen name: ETHANE-PROPANE

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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: 2400 nm / Nominal defocus min: 600 nm
Specimen holderCryogen: NITROGEN
Image recordingElectron dose: 72 e/Å2 / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

EM software
IDNameVersionCategory
7Cootmodel fitting
9RELION3.1model refinement
12RELION3.1classification
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.04 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 233072 / Symmetry type: POINT
Atomic model buildingSpace: REAL
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00414706
ELECTRON MICROSCOPYf_angle_d0.61919926
ELECTRON MICROSCOPYf_dihedral_angle_d3.8072000
ELECTRON MICROSCOPYf_chiral_restr0.0412246
ELECTRON MICROSCOPYf_plane_restr0.0042510

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