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- PDB-8bw7: Cryo-EM structure of rat SLC22A6 bound to alpha-ketoglutaric acid -

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Basic information

Entry
Database: PDB / ID: 8bw7
TitleCryo-EM structure of rat SLC22A6 bound to alpha-ketoglutaric acid
Components
  • Solute carrier family 22 member 6
  • Synthetic nanobody (Sybody)
KeywordsMEMBRANE PROTEIN / transporter
Function / homology
Function and homology information


Organic anion transport / renal tubular secretion / alpha-ketoglutarate transmembrane transporter activity / alpha-ketoglutarate transport / sodium-independent organic anion transmembrane transporter activity / sodium-independent organic anion transport / metanephric proximal tubule development / prostaglandin transport / prostaglandin transmembrane transporter activity / organic anion transport ...Organic anion transport / renal tubular secretion / alpha-ketoglutarate transmembrane transporter activity / alpha-ketoglutarate transport / sodium-independent organic anion transmembrane transporter activity / sodium-independent organic anion transport / metanephric proximal tubule development / prostaglandin transport / prostaglandin transmembrane transporter activity / organic anion transport / organic anion transmembrane transporter activity / solute:inorganic anion antiporter activity / monoatomic anion transport / antiporter activity / chloride ion binding / xenobiotic transmembrane transporter activity / transmembrane transporter activity / basal plasma membrane / caveola / response to organic cyclic compound / basolateral plasma membrane / protein-containing complex / identical protein binding / plasma membrane
Similarity search - Function
Organic cation transport protein/SVOP / Major facilitator, sugar transporter-like / Sugar (and other) transporter / Major facilitator superfamily domain / Major facilitator superfamily (MFS) profile. / MFS transporter superfamily
Similarity search - Domain/homology
2-OXOGLUTARIC ACID / Solute carrier family 22 member 6
Similarity search - Component
Biological speciesRattus norvegicus (Norway rat)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.53 Å
AuthorsParker, J.L. / Kato, T. / Newstead, S.
Funding support United Kingdom, 3items
OrganizationGrant numberCountry
Wellcome Trust215519 United Kingdom
Wellcome Trust219531 United Kingdom
Medical Research Council (MRC, United Kingdom)MR/S021043/1 United Kingdom
CitationJournal: Nat Struct Mol Biol / Year: 2023
Title: Molecular basis for selective uptake and elimination of organic anions in the kidney by OAT1.
Authors: Joanne L Parker / Takafumi Kato / Gabriel Kuteyi / Oleg Sitsel / Simon Newstead /
Abstract: In mammals, the kidney plays an essential role in maintaining blood homeostasis through the selective uptake, retention or elimination of toxins, drugs and metabolites. Organic anion transporters ...In mammals, the kidney plays an essential role in maintaining blood homeostasis through the selective uptake, retention or elimination of toxins, drugs and metabolites. Organic anion transporters (OATs) are responsible for the recognition of metabolites and toxins in the nephron and their eventual urinary excretion. Inhibition of OATs is used therapeutically to improve drug efficacy and reduce nephrotoxicity. The founding member of the renal organic anion transporter family, OAT1 (also known as SLC22A6), uses the export of α-ketoglutarate (α-KG), a key intermediate in the Krebs cycle, to drive selective transport and is allosterically regulated by intracellular chloride. However, the mechanisms linking metabolite cycling, drug transport and intracellular chloride remain obscure. Here, we present cryogenic-electron microscopy structures of OAT1 bound to α-KG, the antiviral tenofovir and clinical inhibitor probenecid, used in the treatment of Gout. Complementary in vivo cellular assays explain the molecular basis for α-KG driven drug elimination and the allosteric regulation of organic anion transport in the kidney by chloride.
History
DepositionDec 6, 2022Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jul 19, 2023Provider: repository / Type: Initial release
Revision 1.1Aug 2, 2023Group: Database references / Category: citation / citation_author / Item: _citation.pdbx_database_id_PubMed / _citation.title
Revision 1.2Nov 22, 2023Group: Data collection / Database references
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation_author.identifier_ORCID

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Solute carrier family 22 member 6
B: Synthetic nanobody (Sybody)
hetero molecules


Theoretical massNumber of molelcules
Total (without water)76,8154
Polymers76,6332
Non-polymers1822
Water0
1
A: Solute carrier family 22 member 6
hetero molecules


Theoretical massNumber of molelcules
Total (without water)60,7353
Polymers60,5541
Non-polymers1822
Water0
TypeNameSymmetry operationNumber
identity operation1_5551
2
B: Synthetic nanobody (Sybody)


Theoretical massNumber of molelcules
Total (without water)16,0801
Polymers16,0801
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Solute carrier family 22 member 6 / Organic anion transporter 1 / rOAT1 / renal organic anion transporter 1 / rROAT1


Mass: 60553.559 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Rattus norvegicus (Norway rat) / Gene: Slc22a6, Oat1 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: O35956
#2: Antibody Synthetic nanobody (Sybody)


Mass: 16079.819 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) synthetic construct (others) / Production host: Escherichia coli (E. coli)
#3: Chemical ChemComp-AKG / 2-OXOGLUTARIC ACID / Α-Ketoglutaric acid


Mass: 146.098 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C5H6O5 / Feature type: SUBJECT OF INVESTIGATION
#4: Chemical ChemComp-CL / CHLORIDE ION / Chloride


Mass: 35.453 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Cl / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY

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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: OAT1 / Type: COMPLEX / Entity ID: #1-#2 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Rattus norvegicus (Norway rat)
Source (recombinant)Organism: Spodoptera frugiperda (fall armyworm)
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationCryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K

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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 / Nominal defocus max: 2000 nm / Nominal defocus min: 800 nm
Image recordingElectron dose: 42 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)
EM imaging opticsEnergyfilter name: GIF Bioquantum

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Processing

Software
NameVersionClassification
phenix.real_space_refine1.20.1_4487refinement
PHENIX1.20.1_4487refinement
EM software
IDNameVersionCategory
2EPUimage acquisition
7UCSF Chimeramodel fitting
12cryoSPARC3.3.23D reconstruction
13PHENIX1.20.1model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.53 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 240196 / Symmetry type: POINT
Atomic model buildingSpace: REAL
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 81.47 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00254860
ELECTRON MICROSCOPYf_angle_d0.57236606
ELECTRON MICROSCOPYf_chiral_restr0.0387755
ELECTRON MICROSCOPYf_plane_restr0.0042826
ELECTRON MICROSCOPYf_dihedral_angle_d4.2068672

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