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Molecular basis of polyspecific drug and xenobiotic recognition by OCT1 and OCT2

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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 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.

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Fig. 1: Cryo-EM structures of OCTs 1 and 2.
Fig. 2: DPH recognition by OCT1.
Fig. 3: VPM recognition by OCT1.
Fig. 4: General principles of organic cation recognition by OCT1.
Fig. 5: Extracellular gate closure in OCTs.

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Data availability

Atomic coordinates have been deposited in the Protein Data Bank with the PDB IDs 8ET6 (Apo-OCT1CS), 8ET7 (DPH-OCT1CS) and 8ET8 (VPM-OCT1CS), 8ET9 (MPP+-OCT2CS), respectively. The reconstructed cryo-EM maps have been deposited in the Electron Microscopy Data Bank with the IDs EMD-28586 (Apo-OCT1CS), EMD-28587 (DPH-OCT1CS) and EMD-28588 (VPM-OCT1CS), EMD-28589 (MPP+-OCT2CS), respectively. Source data are provided with this paper. Additional data pertinent to this paper are available upon reasonable request to S.-Y.L.

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Acknowledgements

Cryo-EM data were screened and collected at the Duke University Shared Materials Instrumentation Facility (SMIF), the UNC Cryo-EM core facility and the National Institute of Environmental Health Sciences (NIEHS). We thank N. Bhattacharya at SMIF, and J. Strauss of the UNC Cryo-EM Core Facility for assistance with the microscope operation. This research was supported by a National Institutes of Health (R01GM137421 to S.-Y.L. and R01GM138472 to W.I.), the National Institute of Health Intramural Research Program; US National Institutes of Environmental Health Science (ZIC ES103326 to M.J.B.) and a National Science Foundation grant MCB-1810695 (W.I.). DUKE SMIF is affiliated with the North Carolina Research Triangle Nanotechnology Network, which is in part supported by the NSF (ECCS-2025064). The UNC CryoEM core facility is supported by NIH grant P30CA016086.

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Author notes

  1. These authors contributed equally: Yang Suo, Nicholas J. Wright.

Authors and Affiliations

  1. Department of Biochemistry, Duke University School of Medicine, Durham, NC, USA

    Yang Suo, Nicholas J. Wright, Justin G. Fedor & Seok-Yong Lee

  2. Departments of Biological Sciences, Chemistry, and Bioengineering, Lehigh University, Bethlehem, PA, USA

    Hugo Guterres & Wonpil Im

  3. Genome Integrity and Structural Biology Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, Department of Health and Human Services, Research Triangle Park, Durham, NC, USA

    Kevin John Butay & Mario J. Borgnia

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  1. Yang Suo
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Contributions

Y.S. conducted biochemical preparation, sample freezing, grid screening, data collection, data processing and single particle 3D reconstruction as well as surface expression experiments, N.J.W. performed radiotracer uptake assays, data processing and single particle 3D reconstruction, all under the guidance of S.-Y.L. J.G.F. performed part of radiotracer uptake and surface expression experiments. N.J.W., Y.S. and S.-Y.L. performed model building and refinement. H.G. carried out all MD simulations as well as docking studies under the guidance of W.I. K.J.B. helped with part of cryo-EM sample screening and provided advice on sample freezing under the guidance of M.J.B. N.J.W., Y.S. and S.-Y.L. wrote the paper.

Corresponding author

Correspondence to Seok-Yong Lee.

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The authors declare no competing interests.

Peer review

Peer review information

Nature Structural & Molecular Biology thanks Cornelius Gati and Mladen V. Tzvetkov for their contribution to the peer review of this work. Primary Handling Editor: Katarzyna Ciazynska, in collaboration with the Nature Structural & Molecular Biology team. Peer reviewer reports are available.

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Extended data

Extended Data Fig. 1 Consensus mutagenesis, protein biochemistry, and cryo-EM analysis of OCT1CS.

a, FSEC traces showing strong monodisperse peaks for OCT1CS-GFP and OCT2CS-GFP, while WT hOCT1-GFP and WT hOCT2-GFP transfected HEK293T cells did not yield any discernable peak corresponding to target protein. Asterisk indicates target protein peak in FSEC. b, Map of all residues in OCT1CS and OCT2CS that deviate from WT hOCT1. The residues are colored based on their conservation score from MAFFT alignment. Blue spheres indicate mildly changed, while red spheres indicate drastic changes. c, Time-dependent accumulation of 10 nM [3H]-MPP+ in WT hOCT1 and OCT1CS expressing oocytes (n = 3 biologically independent replicates per timepoint). d, Raw uptake values for controls in the OCT1 [14C]-metformin uptake experiments, corresponding to Fig. 1e (n = 3–4 biologically independent replicates, shown with mean ± s.e.m.). e, Raw uptake values for controls in the OCT2 [3H]-MPP+ uptake experiments, corresponding to Fig. 1f (n = 3 biologically independent replicates, shown with mean ± s.e.m.). f, [3H]-MPP+ uptake in oocytes expressing Y36C, F446I, and Y36C/F446I in the OCT1CS-GFP background (n = 3 biologically independent replicates, individual values and mean ± S.E.M; water injected controls used for background correction, OCT1CS uptake signal used for normalization). g, FSEC traces showing expression of selected OCT1CS mutants in HEK293T cells. Asterisk indicates target protein peak in FSEC. h, Representative size-exclusion chromatography trace (left) and SDS-PAGE (right) of purified OCT1CS or OCT2CS samples used for cryo-EM grid preparation. The experiments were repeated independently for >10 times with similar results. Asterisks indicate target protein peak (in SEC) and band (in SDS-PAGE). i, Representative micrograph of a OCT1CS sample. OCT2 CS behaves similarly on cryo-EM grids. j, Representative 2D classes from a OCT1CS dataset. k, Secondary structure topology of OCT1 and OCT2.

Source data

Extended Data Fig. 2 Cryo-EM data processing workflow.

a-d, cryo-EM data processing workflow for apo-OCT1CS, DPH-OCT1CS, VPM-OCT1CS, and MPP+-OCT2CS datasets, respectively.

Extended Data Fig. 3 Cryo-EM data validation.

a, Final cryo-EM reconstructions. b, Fourier-shell correlation for the final reconstruction, generated from cryoSPARC. c, projection orientation distribution map for the final reconstruction, generated from cryoSPARC. d, Map-to-model correlation plots. e, Local Resolution plots. f, cryo-EM maps for secondary structure segments. From left to right are cryo-EM data validations for apo-OCT1CS, DPH-OCT1CS, VPM-OCT1CS, and MPP+-OCT2CS datasets, respectively.

Extended Data Fig. 4 Validation of ligand binding poses with molecular dynamics simulations.

a, Three possible poses for DPH molecule placement based on the cryo-EM reconstruction. b, Final MD frame for 5 replicas of DPH-OCT1CS MD simulations (500 ns) for the three proposed poses, where possibility #1 is more stably bound at the site. c, Two possible poses for S(–)-VPM based on the cryo-EM reconstruction. d, Final MD frame for 5 replicas of VPM-OCT1CS MD simulations (500 ns), for the two proposed poses, where possibility #2 is more stable (r.m.s.d. – root mean square deviation from starting pose). e, Zoom-in view of the cryo-EM map and model of the VPM chiral center. f, Inter-atomic distances between the ionizable nitrogen of VPM or DPH and acidic residues (D474 or E386) during the MD-simulations of drug-bound OCT1CS (scatter plot showing individual values extracted per MD frame, compiled from all 5 replicas per condition, with the black line representing the mean).

Source data

Extended Data Fig. 5 Surface expression of hOCT1-WT, OCT1CS and mutants.

Representative confocal microscopy images showing surface expression of OCT1CS and relevant mutants in Xenopus laevis oocytes used for radiotracer uptake studies. Scale bars represent 200 mm. Similar results were observed in 6–8 additional biological replicates per condition.

Extended Data Fig. 6 Ligand-induced local conformational changes in OCT1CS.

a, Structural overlay of apo-OCT1CS (marine), VPM-OCT1CS (green) and DPH-OCT1CS (yellow), showing that no large conformational changes are present among the three structures. While other residues remain relatively stable, Y36 exhibits considerable rotamer movement among the three structures.

Extended Data Fig. 7 In silico ligand docking.

In-silico docking and short-time scale (50 ns) MD simulations for serotonin, epinephrine, metformin, dopamine, mescaline, norfentanyl, methylnaltrexone, morphine, imipramine and MPP+, respectively. For each ligand, Top MMPBSA scored poses are shown in the large panels, with other candidate poses (under 3 Å ligand r.m.s.d. at the conclusion of the simulation) shown below. Self-docking runs of DPH and VPM shown at top left for validation. PDBs corresponding to all poses shown here are available as Source Data.

Extended Data Fig. 8 Local conformational changes associated with OCT gating.

a, ConSurf plot for OCT2cs and OCT2 homologs. b, Electrostatics surface of outward occluded OCT2, calculated by APBS. c, Concerted local conformational changes in TM2 and 11 leads to extracellular gate formation. d, Local conformational changes in the N-lobe from outward open (blue) to outward occluded (green) conformations.

Extended Data Table 1 MMPBSA scores for top in silico docking poses
Full size table

Supplementary information

Supplementary Information

Supplementary Fig. 1. Multiple sequence alignment. Multiple sequence alignment of OCT1CS, human OCTs (SLC22A1-3) and representative human OATs (SLC22A7-9). Sequences are aligned using MAFFT57. E386 and D474 (numbering according to hOCT1) positions are highlighted in red and 217, 244, 354 and 446 (numbering according to hOCT1) in green.

Reporting Summary

Peer Review File

Supplementary Data 1

Compressed (.zip) folder containing final MD frame PDB files from all poses from the Docking/MD to OCT1CS. Excel file included in folder containing all file names and pertinent information.

Source data

Source Data Fig. 1

Source data for Fig. 1.

Source Data Fig. 2

Source data for Fig. 2.

Source Data Fig. 3

Source data for Fig. 3.

Source Data Fig. 4

Source data for Fig. 4.

Source Data Extended Data Fig. 1

Uncropped gel images for Extended Data Fig. 1h.

Source Data Extended Data Fig. 1

Source data for Extended Data Fig. 1.

Source Data Extended Data Fig. 4

Source data for Extended Data Fig. 4f.

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Suo, Y., Wright, N.J., Guterres, H. et al. Molecular basis of polyspecific drug and xenobiotic recognition by OCT1 and OCT2. Nat Struct Mol Biol 30, 1001–1011 (2023). https://doi.org/10.1038/s41594-023-01017-4

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