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- PDB-8et9: Cryo-EM structure of the organic cation transporter 2 in complex ... -

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Basic information

Entry
Database: PDB / ID: 8et9
TitleCryo-EM structure of the organic cation transporter 2 in complex with 1-methyl-4-phenylpyridinium
ComponentsOCT2
KeywordsTRANSPORT PROTEIN / Cation / transport / membrane protein
Function / homology1-methyl-4-phenylpyridin-1-ium
Function and homology information
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.61 Å
AuthorsSuo, Y. / Wright, N.J. / Lee, S.-Y.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM137421 United States
Citation
Journal: Nat Struct Mol Biol / Year: 2023
Title: Molecular basis of polyspecific drug and xenobiotic recognition by OCT1 and OCT2.
Authors: Yang Suo / Nicholas J Wright / Hugo Guterres / Justin G Fedor / Kevin John Butay / Mario J Borgnia / Wonpil Im / Seok-Yong Lee /
Abstract: A wide range of endogenous and xenobiotic organic ions require facilitated transport systems to cross the plasma membrane for their disposition. In mammals, organic cation transporter (OCT) subtypes ...A wide range of endogenous and xenobiotic organic ions require facilitated transport systems to cross the plasma membrane for their disposition. In mammals, organic cation transporter (OCT) subtypes 1 and 2 (OCT1 and OCT2, also known as SLC22A1 and SLC22A2, respectively) are polyspecific transporters responsible for the uptake and clearance of structurally diverse cationic compounds in the liver and kidneys, respectively. Notably, it is well established that human OCT1 and OCT2 play central roles in the pharmacokinetics and drug-drug interactions of many prescription medications, including metformin. Despite their importance, the basis of polyspecific cationic drug recognition and the alternating access mechanism for OCTs have remained a mystery. Here we present four cryo-electron microscopy structures of apo, substrate-bound and drug-bound OCT1 and OCT2 consensus variants, in outward-facing and outward-occluded states. Together with functional experiments, in silico docking and molecular dynamics simulations, these structures uncover general principles of organic cation recognition by OCTs and provide insights into extracellular gate occlusion. Our findings set the stage for a comprehensive structure-based understanding of OCT-mediated drug-drug interactions, which will prove critical in the preclinical evaluation of emerging therapeutics.
#1: Journal: bioRxiv / Year: 2023
Title: Molecular basis of polyspecific drug binding and transport by OCT1 and OCT2.
Authors: Yang Suo / Nicholas J Wright / Hugo Guterres / Justin G Fedor / Kevin John Butay / Mario J Borgnia / Wonpil Im / Seok-Yong Lee /
Abstract: A wide range of endogenous and xenobiotic organic ions require facilitated transport systems to cross the plasma membrane for their disposition . In mammals, organic cation transporter subtypes 1 ...A wide range of endogenous and xenobiotic organic ions require facilitated transport systems to cross the plasma membrane for their disposition . In mammals, organic cation transporter subtypes 1 and 2 (OCT1 and OCT2, also known as SLC22A1 and SLC22A2, respectively) are polyspecific transporters responsible for the uptake and clearance of structurally diverse cationic compounds in the liver and kidneys, respectively . Notably, it is well established that human OCT1 and OCT2 play central roles in the pharmacokinetics, pharmacodynamics, and drug-drug interactions (DDI) of many prescription medications, including metformin . Despite their importance, the basis of polyspecific cationic drug recognition and the alternating access mechanism for OCTs have remained a mystery. Here, we present four cryo-EM structures of apo, substrate-bound, and drug-bound OCT1 and OCT2 in outward-facing and outward-occluded states. Together with functional experiments, docking, and molecular dynamics simulations, these structures uncover general principles of organic cation recognition by OCTs and illuminate unexpected features of the OCT alternating access mechanism. Our findings set the stage for a comprehensive structure-based understanding of OCT-mediated DDI, which will prove critical in the preclinical evaluation of emerging therapeutics.
History
DepositionOct 16, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0May 31, 2023Provider: repository / Type: Initial release
Revision 1.1Jul 12, 2023Group: Database references / Category: citation / citation_author
Revision 1.2Aug 2, 2023Group: Database references / Category: 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: OCT2
hetero molecules


Theoretical massNumber of molelcules
Total (without water)62,2012
Polymers62,0311
Non-polymers1701
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein OCT2


Mass: 62030.547 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Production host: Homo sapiens (human)
#2: Chemical ChemComp-WRF / 1-methyl-4-phenylpyridin-1-ium


Mass: 170.230 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C12H12N / 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: OCT2 / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 60 kDa/nm / Experimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Homo sapiens (human)
Buffer solutionpH: 8
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMTrisTris1
2150 mMSodium ChlorideNaClSodium chloride1
30.06 %DigitoninDigitonin1
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: UltrAuFoil R1.2/1.3
VitrificationInstrument: LEICA EM GP / Cryogen name: ETHANE / Humidity: 90 % / 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 magnification: 81000 X / Nominal defocus max: 2000 nm / Nominal defocus min: 800 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm / Alignment procedure: ZEMLIN TABLEAU
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 3.7 sec. / Electron dose: 60 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 8029
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV
Image scansWidth: 5760 / Height: 4092

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Processing

SoftwareName: PHENIX / Version: 1.19.2_4158: / Classification: refinement
EM software
IDNameVersionCategory
1cryoSPARC3.3particle selection
2Latitude3image acquisition
4Gctf1.18CTF correction
7Coot0.9model fitting
9cryoSPARC3.2initial Euler assignment
10cryoSPARC3.2final Euler assignment
11cryoSPARC3.2classification
12cryoSPARC3.23D reconstruction
13PHENIX1.19model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1141906
3D reconstructionResolution: 3.61 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 73474 / Algorithm: FOURIER SPACE / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingB value: 141.9 / Protocol: AB INITIO MODEL / Space: REAL
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0023994
ELECTRON MICROSCOPYf_angle_d0.4935456
ELECTRON MICROSCOPYf_dihedral_angle_d10.6091347
ELECTRON MICROSCOPYf_chiral_restr0.035634
ELECTRON MICROSCOPYf_plane_restr0.003678

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