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- EMDB-17537: Structure of 5D3-Fab and nanobody(Nb8)-bound ABCG2 -

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Basic information

Entry
Database: EMDB / ID: EMD-17537
TitleStructure of 5D3-Fab and nanobody(Nb8)-bound ABCG2
Map data
Sample
  • Complex: Structure of nanobody (Nb8)-bound ABCG2
    • Complex: ATP-binding cassette sub-family G member 2
      • Protein or peptide: ATP-binding cassette sub-family G member 2
    • Complex: NanobodySingle-domain antibody
      • Protein or peptide: NanobodySingle-domain antibody
    • Complex: 5D3(Fab) heavy and light chain variable domain
      • Protein or peptide: 5D3(Fab) heavy chain variable domain
      • Protein or peptide: 5D3(Fab) light chain variable domain
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)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.04 Å
AuthorsIrobalieva RN / Manolaridis I / Jackson SM / Ni D / Pardon E / Stahlberg H / Steyaert J / Locher KP
Funding support Switzerland, 1 items
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, 2023-
Header (metadata) releaseAug 30, 2023-
Map releaseAug 30, 2023-
UpdateSep 13, 2023-
Current statusSep 13, 2023Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_17537.png

Downloads & links

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Map

FileDownload / File: emd_17537.map.gz / Format: CCP4 / Size: 216 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.84 Å/pix.
x 384 pix.
= 322.56 Å		0.84 Å/pix.
x 384 pix.
= 322.56 Å		0.84 Å/pix.
x 384 pix.
= 322.56 Å

Surface

Projections

Slices (1/3)

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Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.84 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.00402
Minimum - Maximum-0.023524502 - 0.032522883
Average (Standard dev.)0.000010928011 (±0.00061829994)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions384384384
Spacing384384384
CellA=B=C: 322.56 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_17537_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_17537_half_map_2.map
Projections & Slices
AxesZYX

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Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Structure of nanobody (Nb8)-bound ABCG2

EntireName: Structure of nanobody (Nb8)-bound ABCG2
Components
  • Complex: Structure of nanobody (Nb8)-bound ABCG2
    • Complex: ATP-binding cassette sub-family G member 2
      • Protein or peptide: ATP-binding cassette sub-family G member 2
    • Complex: NanobodySingle-domain antibody
      • Protein or peptide: NanobodySingle-domain antibody
    • Complex: 5D3(Fab) heavy and light chain variable domain
      • Protein or peptide: 5D3(Fab) heavy chain variable domain
      • Protein or peptide: 5D3(Fab) light chain variable domain

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Supramolecule #1: Structure of nanobody (Nb8)-bound ABCG2

SupramoleculeName: Structure of nanobody (Nb8)-bound ABCG2 / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all

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Supramolecule #2: ATP-binding cassette sub-family G member 2

SupramoleculeName: ATP-binding cassette sub-family G member 2 / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1
Source (natural)Organism: Homo sapiens (human)

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Supramolecule #3: Nanobody

SupramoleculeName: Nanobody / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2
Source (natural)Organism: Lama glama (llama)

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Supramolecule #4: 5D3(Fab) heavy and light chain variable domain

SupramoleculeName: 5D3(Fab) heavy and light chain variable domain / type: complex / ID: 4 / Parent: 1 / Macromolecule list: #3-#4
Source (natural)Organism: Mus musculus (house mouse)

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Macromolecule #1: ATP-binding cassette sub-family G member 2

MacromoleculeName: ATP-binding cassette sub-family G member 2 / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO / EC number: ABC-type xenobiotic transporter
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 72.385852 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MSSSNVEVFI PVSQGNTNGF PATASNDLKA FTEGAVLSFH NICYRVKLKS GFLPCRKPVE KEILSNINGI MKPGLNAILG PTGGGKSSL LDVLAARKDP SGLSGDVLIN GAPRPANFKC NSGYVVQDDV VMGTLTVREN LQFSAALRLA TTMTNHEKNE R INRVIQEL ...String:
MSSSNVEVFI PVSQGNTNGF PATASNDLKA FTEGAVLSFH NICYRVKLKS GFLPCRKPVE KEILSNINGI MKPGLNAILG PTGGGKSSL LDVLAARKDP SGLSGDVLIN GAPRPANFKC NSGYVVQDDV VMGTLTVREN LQFSAALRLA TTMTNHEKNE R INRVIQEL GLDKVADSKV GTQFIRGVSG GERKRTSIGM ELITDPSILF LDEPTTGLDS STANAVLLLL KRMSKQGRTI IF SIHQPRY SIFKLFDSLT LLASGRLMFH GPAQEALGYF ESAGYHCEAY NNPADFFLDI INGDSTAVAL NREEDFKATE IIE PSKQDK PLIEKLAEIY VNSSFYKETK AELHQLSGGE KKKKITVFKE ISYTTSFCHQ LRWVSKRSFK NLLGNPQASI AQII VTVVL GLVIGAIYFG LKNDSTGIQN RAGVLFFLTT NQCFSSVSAV ELFVVEKKLF IHEYISGYYR VSSYFLGKLL SDLLP MRML PSIIFTCIVY FMLGLKPKAD AFFVMMFTLM MVAYSASSMA LAIAAGQSVV SVATLLMTIC FVFMMIFSGL LVNLTT IAS WLSWLQYFSI PRYGFTALQH NEFLGQNFCP GLNATGNNPC NYATCTGEEY LVKQGIDLSP WGLWKNHVAL ACMIVIF LT IAYLKLLFLK KYS

UniProtKB: Broad substrate specificity ATP-binding cassette transporter ABCG2

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Macromolecule #2: Nanobody

MacromoleculeName: Nanobody / type: protein_or_peptide / ID: 2 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Lama glama (llama)
Molecular weightTheoretical: 13.481859 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
QVQLQESGGG LVQPGGSLRL SCAASGFTLG YYAVGWFRQA PGKEREGVSC ISSSDGSTYY PDSVKGRFTI SRDNAKNTVY LQMNSLKPE DTAVYYCATT VRINNSCKEY QYHGWGQGTQ VTVSS

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Macromolecule #3: 5D3(Fab) heavy chain variable domain

MacromoleculeName: 5D3(Fab) heavy chain variable domain / type: protein_or_peptide / ID: 3 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 23.843633 KDa
Recombinant expressionOrganism: Mus musculus (house mouse)
SequenceString: QVQLQESGPG LVKPSQSLSL TCTVTGFSIT SDYAWNWIRQ FPGKKLEWMG YINFDGGTTY NPSLRGRISI TRDTSKNQFF LQLRSVTPE DTATYYCATF YGAKGTLDYW GQGTSVTVSS AKTTPPSVYP LAPVCGDTSG SSVTLGCLVK GYFPEPVTLT W NSGSLSSG ...String:
QVQLQESGPG LVKPSQSLSL TCTVTGFSIT SDYAWNWIRQ FPGKKLEWMG YINFDGGTTY NPSLRGRISI TRDTSKNQFF LQLRSVTPE DTATYYCATF YGAKGTLDYW GQGTSVTVSS AKTTPPSVYP LAPVCGDTSG SSVTLGCLVK GYFPEPVTLT W NSGSLSSG VHTFPAVLQS DLYTLSSSVT VTSSTWPSQS ITCNVAHPAS STKVDKKIEP RGP

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Macromolecule #4: 5D3(Fab) light chain variable domain

MacromoleculeName: 5D3(Fab) light chain variable domain / type: protein_or_peptide / ID: 4 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 23.594016 KDa
Recombinant expressionOrganism: Mus musculus (house mouse)
SequenceString: DIVLTQSPSS FSVSLGDRVT ISCKASGYIL NRLAWYQQKP GNAPRLLISG ATSLETGFPS RFSGTGSGKD YTLSISSLQT EDVGTYYCQ QYWSTPWTFG GGTKLEIRRA DAAPTVSIFP PSSEQLTSGG ASVVCFLNNF YPKDINVKWK IDGSERQNGV L NSWTDQDS ...String:
DIVLTQSPSS FSVSLGDRVT ISCKASGYIL NRLAWYQQKP GNAPRLLISG ATSLETGFPS RFSGTGSGKD YTLSISSLQT EDVGTYYCQ QYWSTPWTFG GGTKLEIRRA DAAPTVSIFP PSSEQLTSGG ASVVCFLNNF YPKDINVKWK IDGSERQNGV L NSWTDQDS KDSTYSMSST LTLTKDEYER HNSYTCEATH KTSTSPIVKS FNRNEC

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration1 mg/mL
BufferpH: 7.5
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE-PROPANE

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Electron microscopy

MicroscopeTFS KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 2.4 µm / Nominal defocus min: 0.6 µm
Sample stageCooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Average electron dose: 72.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: NONE
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.04 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 233072
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: REAL
Output model

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

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