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- PDB-8qcm: ABCG2 in complex with MZ82 and 5D3 Fab -

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Basic information

Entry
Database: PDB / ID: 8qcm
TitleABCG2 in complex with MZ82 and 5D3 Fab
Components
  • 5D3(Fab) heavy chain variable domain
  • 5D3(Fab) light chain variable domain
  • Broad substrate specificity ATP-binding cassette transporter ABCG2
KeywordsTRANSPORT PROTEIN / ABC transporter / multidrug resistance
Function / homology
Function and homology information


biotin transmembrane transporter activity / biotin transport / riboflavin transport / riboflavin transmembrane transporter activity / sphingolipid transporter activity / renal urate salt excretion / Abacavir transmembrane transport / urate metabolic process / urate transmembrane transporter activity / sphingolipid biosynthetic process ...biotin transmembrane transporter activity / biotin transport / riboflavin transport / riboflavin transmembrane transporter activity / sphingolipid transporter activity / renal urate salt excretion / Abacavir transmembrane transport / urate metabolic process / urate transmembrane transporter activity / sphingolipid biosynthetic process / Sphingolipid de novo biosynthesis / external side of apical plasma membrane / organic anion transport / xenobiotic transport across blood-brain barrier / : / transepithelial transport / Ciprofloxacin ADME / export across plasma membrane / Paracetamol ADME / NFE2L2 regulating MDR associated enzymes / Differentiation of Keratinocytes in Interfollicular Epidermis in Mammalian Skin / ABC-type xenobiotic transporter / Heme biosynthesis / cellular detoxification / ABC-type xenobiotic transporter activity / Heme degradation / efflux transmembrane transporter activity / xenobiotic transmembrane transporter activity / ATPase-coupled transmembrane transporter activity / transport across blood-brain barrier / Iron uptake and transport / brush border membrane / mitochondrial membrane / transmembrane transport / apical plasma membrane / membrane raft / protein homodimerization activity / ATP hydrolysis 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
CHOLESTEROL / : / Broad substrate specificity ATP-binding cassette transporter ABCG2
Similarity search - Component
Biological speciesHomo sapiens (human)
Mus musculus (house mouse)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.39 Å
AuthorsYu, Q. / Kowal, J. / Ni, D. / Stahlberg, H. / Tajkhorshid, E. / Altmann, K.H. / Locher, K.P.
Funding support Switzerland, 1items
OrganizationGrant numberCountry
Swiss National Science Foundationnot applicable Switzerland
CitationJournal: ACS Chem Biol / Year: 2024
Title: Modulation of ABCG2 Transporter Activity by Ko143 Derivatives.
Authors: Qin Yu / Sepehr Dehghani-Ghahnaviyeh / Ali Rasouli / Anna Sadurni / Julia Kowal / Rose Bang-Soerensen / Po-Chao Wen / Melanie Tinzl-Zechner / Rossitza N Irobalieva / Dongchun Ni / Henning ...Authors: Qin Yu / Sepehr Dehghani-Ghahnaviyeh / Ali Rasouli / Anna Sadurni / Julia Kowal / Rose Bang-Soerensen / Po-Chao Wen / Melanie Tinzl-Zechner / Rossitza N Irobalieva / Dongchun Ni / Henning Stahlberg / Karl-Heinz Altmann / Emad Tajkhorshid / Kaspar P Locher /
Abstract: ABCG2 is a multidrug transporter that protects tissues from xenobiotics, affects drug pharmacokinetics, and contributes to multidrug resistance of cancer cells. Here, we present tetracyclic ...ABCG2 is a multidrug transporter that protects tissues from xenobiotics, affects drug pharmacokinetics, and contributes to multidrug resistance of cancer cells. Here, we present tetracyclic fumitremorgin C analog Ko143 derivatives, evaluate their modulation of purified ABCG2, and report four high-resolution cryo-EM structures and computational analyses to elucidate their interactions with ABCG2. We found that Ko143 derivatives that are based on a ring-opened scaffold no longer inhibit ABCG2-mediated transport activity. In contrast, closed-ring, tetracyclic analogs were highly potent inhibitors. Strikingly, the least potent of these compounds, MZ82, bound deeper into the central ABCG2 cavity than the other inhibitors and it led to partial closure of the transmembrane domains and increased flexibility of the nucleotide-binding domains. Minor structural modifications can thus convert a potent inhibitor into a compound that induces conformational changes in ABCG2 similar to those observed during binding of a substrate. Molecular dynamics simulations and free energy binding calculations further supported the correlation between reduced potency and distinct binding pose of the compounds. We introduce the highly potent inhibitor AZ99 that may exhibit improved stability.
History
DepositionAug 27, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0Nov 6, 2024Provider: repository / Type: Initial release
Revision 1.1Nov 27, 2024Group: Data collection / Database references / Category: citation / citation_author / em_admin
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation_author.identifier_ORCID / _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Broad substrate specificity ATP-binding cassette transporter ABCG2
C: 5D3(Fab) light chain variable domain
D: 5D3(Fab) heavy chain variable domain
E: 5D3(Fab) light chain variable domain
F: 5D3(Fab) heavy chain variable domain
B: Broad substrate specificity ATP-binding cassette transporter ABCG2
hetero molecules


Theoretical massNumber of molelcules
Total (without water)245,96116
Polymers241,9296
Non-polymers4,03210
Water95553
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails
d_1ens_1chain "A"
d_2ens_1chain "B"
d_1ens_2chain "E"
d_2ens_2chain "C"
d_1ens_3chain "F"
d_2ens_3chain "D"

NCS domain segments:

Component-ID: 1

Dom-IDEns-IDBeg auth comp-IDBeg label comp-IDEnd auth comp-IDEnd label comp-IDAuth asym-IDLabel asym-IDAuth seq-IDLabel seq-ID
d_1ens_1GLYGLYTYRTYRAA34 - 65444 - 664
d_2ens_1GLYGLYTYRTYRBF34 - 65444 - 664
d_1ens_2ASPASPARGARGED1 - 1071 - 107
d_2ens_2ASPASPARGARGCB1 - 1071 - 107
d_1ens_3VALVALSERSERFE2 - 1192 - 119
d_2ens_3VALVALSERSERDC2 - 1192 - 119

NCS ensembles :
ID
ens_1
ens_2
ens_3

NCS oper:
IDCodeMatrixVector
1given(-0.999712633719, 0.0136399025059, -0.0197130171014), (-0.014768259603, -0.998191303368, 0.0582753840802), (-0.0188824916764, 0.0585497646539, 0.998105894466)100.987449099, 98.2674063026, -1.75283527495
2given(-0.999319036744, -0.000392198762311, -0.0368959209214), (-0.0028151872768, -0.99621842598, 0.0868384848883), (-0.0367904542117, 0.086883219998, 0.995538933725)102.654687292, 96.2400835629, -2.24581178716
3given(-0.999407801266, -0.00795369807612, -0.0334781340003), (0.00501969228788, -0.996210671438, 0.086827995501), (-0.0340418780117, 0.0866085261409, 0.995660641856)102.782358812, 95.8249380127, -2.39497678952

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Components

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Antibody , 2 types, 4 molecules CEDF

#2: Antibody 5D3(Fab) light chain variable domain


Mass: 23594.016 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Mus musculus (house mouse)
#3: Antibody 5D3(Fab) heavy chain variable domain


Mass: 23843.633 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Mus musculus (house mouse)

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Protein / Sugars , 2 types, 4 molecules AB

#1: Protein Broad substrate specificity ATP-binding cassette transporter ABCG2 / ATP-binding cassette sub-family G member 2 / Breast cancer resistance protein / CDw338 / ...ATP-binding cassette sub-family G member 2 / Breast cancer resistance protein / CDw338 / Mitoxantrone resistance-associated protein / Placenta-specific ATP-binding cassette transporter / Urate exporter


Mass: 73526.938 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) / Strain (production host): HEK293-EBNA
References: UniProt: Q9UNQ0, ABC-type xenobiotic transporter
#4: Polysaccharide 2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose


Type: oligosaccharide / Mass: 424.401 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DGlcpNAcb1-4DGlcpNAcb1-ROHGlycam Condensed SequenceGMML 1.0
WURCS=2.0/1,2,1/[a2122h-1b_1-5_2*NCC/3=O]/1-1/a4-b1WURCSPDB2Glycan 1.1.0
[][D-1-deoxy-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{}}LINUCSPDB-CARE

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Non-polymers , 3 types, 61 molecules

#5: Chemical ChemComp-V0U / (2~{S},5~{S},8~{S})-14-methoxy-2-(2-methylpropyl)-5-(phenylmethyl)-3,6,17-triazatetracyclo[8.7.0.0^{3,8}.0^{11,16}]heptadeca-1(10),11,13,15-tetraene-4,7-dione


Mass: 431.527 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C26H29N3O3 / Feature type: SUBJECT OF INVESTIGATION
#6: Chemical
ChemComp-CLR / CHOLESTEROL


Mass: 386.654 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: C27H46O
#7: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 53 / Source method: isolated from a natural source / Formula: H2O

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Details

Has ligand of interestY
Has protein modificationY

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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: ABCG2 in complex with MZ82 and 5D3 Fab / Type: COMPLEX
Details: Nanodisc-reconstituted 5D3-Fab-ABCG2 was incubated with 30 uM MZ82
Entity ID: #1-#3 / Source: RECOMBINANT
Molecular weightValue: 0.244 MDa / Experimental value: YES
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Homo sapiens (human) / Cell: HEK293EBNA
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
10.15 MNaCl1
20.04 MHepes1
SpecimenConc.: 0.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: The sample was mono-disperse.
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE-PROPANE / Humidity: 100 % / Chamber temperature: 277 K / Details: Grids were blotted for 2.5s with blot force 1

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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 FIELD / Nominal magnification: 130000 X / Nominal defocus max: 2200 nm / Nominal defocus min: 800 nm / Cs: 2.7 mm / C2 aperture diameter: 100 µm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 42 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV

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Processing

EM software
IDNameVersionCategory
2EPU2image acquisition
4cryoSPARC3CTF correction
7UCSF Chimeramodel fitting
10cryoSPARC3final Euler assignment
11cryoSPARC3classification
12cryoSPARC33D reconstruction
13PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 2.39 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 601814 / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT
Atomic model buildingPDB-ID: 6ffc

6ffc


Accession code: 6ffc / Source name: PDB / Type: experimental model
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 68.39 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00712900
ELECTRON MICROSCOPYf_angle_d0.674317524
ELECTRON MICROSCOPYf_chiral_restr0.04542016
ELECTRON MICROSCOPYf_plane_restr0.00422144
ELECTRON MICROSCOPYf_dihedral_angle_d13.39754582
Refine LS restraints NCS
Ens-IDDom-IDAsym-IDAuth asym-IDRefine-IDTypeRms dev position (Å)
ens_1d_2AAELECTRON MICROSCOPYNCS constraints0.000708181568111
ens_2d_2DEELECTRON MICROSCOPYNCS constraints0.00070230007994
ens_3d_2EFELECTRON MICROSCOPYNCS constraints0.000706861271813

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