Folates are essential nutrients with important roles as cofactors in one-carbon transfer reactions, being heavily utilized in the synthesis of nucleic acids and the metabolism of amino acids during cell division1,2. Mammals lack de novo folate synthesis pathways and thus rely on folate uptake from the extracellular milieu3. The human reduced folate carrier (hRFC, also known as SLC19A1) is the major importer of folates into the cell1,3, as well as chemotherapeutic agents such as methotrexate4,5,6. As an anion exchanger, RFC couples the import of folates and antifolates to anion export across the cell membrane and it is a major determinant in methotrexate (antifolate) sensitivity, as genetic variants and its depletion result in drug resistance4,5,6,7,8. Despite its importance, the molecular basis of substrate specificity by hRFC remains unclear. Here we present cryo-electron microscopy structures of hRFC in the apo state and captured in complex with methotrexate. Combined with molecular dynamics simulations and functional experiments, our study uncovers key determinants of hRFC transport selectivity among folates and antifolate drugs while shedding light on important features of anion recognition by hRFC.
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Coordinates have been deposited in the Protein Data Bank with IDs 7TX6 (hRFCEM–MTX), 7XT7 (hRFCEM) and 8DEP (apo hRFCEM). The cryo-EM maps have been deposited in the Electron Microscopy Data Bank with IDs EMD-26155 (hRFCEM–MTX), EMD-26156 (hRFCEM) and EMD-27394 (apo hRFCEM). Source data are provided with this paper.
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Cryo-EM data were screened and collected at the Duke University Shared Materials Instrumentation Facility (SMIF) and at the Pacific Northwest Center for Cryo-EM (PNCC) at Oregon Health & Science University (OHSU). We thank N. Bhattacharya at SMIF and J. Myers at the PNCC for assistance with microscope operation. This research was supported by National Institutes of Health grant R01GM137421 (S.-Y.L. and J.H.), American Heart Association fellowship 20PRE35210058 (N.J.W.) and National Science Foundation grant MCB-2111728 (W.I.). A portion of this research was supported by National Institutes of Health grant U24GM129547, performed at the PNCC at OHSU and accessed through EMSL (grid.436923.9), a Department of Energy Office of Science User Facility sponsored by the Office of Biological and Environmental Research. The Duke University SMIF is affiliated with the North Carolina Research Triangle Nanotechnology Network, which is in part supported by the National Science Foundation (ECCS-2025064).
The authors declare no competing interests.
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Extended data figures and tables
Extended Data Fig. 1 Protein biochemistry, NHS-MTX protein modification and cryo-EM analysis of hRFCEM.
a, Topology diagram of hRFCEM used for structural elucidation. b, Representative gel-filtration profile for the final purification step and representative SDS-PAGE analysis (Coomassie stained) of purified protein used for cryo-EM grid preparation. Protein laddering during SDS-PAGE is common for small membrane proteins, with degree of non-specific oligomerization denoted by the number of asterisks (* monomer, ** dimer, *** trimer, **** tetramer). This purification is performed routinely with very similar results, reliably yielding pure and biochemically stable protein sample. c, Characterization of MTX modification of RFC by NHS-MTX. A representative spectral deconvolution of the MTX-RFC UV-vis spectrum into MTX and pure unlabeled RFC yields a labelling ratio of 1.1:1 MTX:RFC (graph prepared in Prism 8). d, Cryo-EM micrograph for hRFCEM sample (of representative quality for all collected cryo-EM data reported in this study) and 2D-classes of hRFCEM.
a, Processing workflow for hRFCEM. b, Phenix and cryoSPARC reported Fourier shell correlations, and particle angular distribution for the final focused map. c, Local resolution analysis. d, Cryo-EM density corresponding to hRFCEM TM1-12 (map threshold = 0.20).
a, Processing workflow for Apo hRFCEM. b, Phenix and cryoSPARC reported Fourier shell correlations, and particle angular distribution for the final map. c, Local resolution analysis. d, Cryo-EM density corresponding to Apo hRFCEM TM1–12 (map threshold = 0.25). e, Structural superposition of the final refined coordinates for apo hRFCEM and hRFCEM f, Overlay of the final cryo-EM reconstructions (sharpened maps) for apo hRFCEM and hRFCEM (final coordinates for apo hRFCEM shown for reference, hRFCEM map resampled relative to the Apo hRFCEM map, with map threshold shown at 0.3). g, Weak, spurious cryo-EM density in the transporter central cavity present in both apo hRFCEM and hRFCEM sharpened maps (Map threshold shown at 0.15 for apo hRFCEM, 0.10 for hRFCEM).
a, Processing workflow for MTX modified hRFCEM b, Fourier shell correlation and particle angular distribution for the final reconstruction c, Local resolution analysis of the final reconstruction at two different map thresholds d, Cryo-EM density corresponding to hRFCEM-MTX TM1–12 (map threshold = 0.2). e, Structural superposition of the final refined coordinates for apo hRFCEM (grey) and hRFCEM-MTX (blue), highlighting apparent ligand induced changes in the TM4 conformational state f, Cryo-EM density corresponding to R133 in the apo hRFCEM and hRFCEM-MTX reconstructions (hRFCEM map resampled relative to the hRFCEM-MTX map, with map threshold shown at 0.1) g, Ribbon depiction of superposed structures.
Mapping of clinically relevant mutations of full-length RFC11,35,36,37,38,39,40,41,42,83,84,85 onto the hRFCEM-MTX structure fall into two general regions. MTX-resistance associated mutations observed in tumor samples (red), in cell lines (green), or observed in both tumors and in cell lines (blue). The putative position of the megaloblastic anemia-associated mutation of ΔPhe212 is shown in purple as the model only extends to residue 211. Other residues not resolved in the structure are listed in parenthesis in the legend.
a, The all-atom molecular dynamics system setup for MTX-bound hRFC embedded in a POPC membrane and solvated with 150 mM KCl (Extended Data Table 3). hRFC is shown in cartoon, the N- and C- terminal domains colored in blue and yellow, respectively. MTX is shown as sticks, in pink. Lipids are depicted as spheres with glycerol-palmitoyl and -oleoyl groups colored gray, phosphates in orange, and choline in green. Red and blue spheres represent Cl− and K+, respectively. b, Timecourse traces for n = 5 replicates for MD simulations of MTX-K411 hRFC (MD system “MTX-LYS”). c, Timecourse traces for n = 5 replicates for MD simulations of hRFC with unlinked MTX (MD system “MTX”). For b and c, distances from the MTX N4 to E123 carboxylate center-of-mass (blue), and the l-Glu center-of-mass of MTX to Arg guanidiniums (R133, red; R157, purple; R373, cyan), are plotted as a function of time. d, Histogram plot of MTX N4 to E123 distances over n = 5 replicates. e. Histogram plots of MTX α- and γ- carboxylates to Arg guanidiniums over n = 5 replicates. f–h, MD simulations of PT523 docked into hRFC. f, Chemical structure of PT523 compared to MTX with the structural difference highlighted in red. g, the cryo-EM structure of MTX-labelled hRFC versus the MD simulation of docked PT523 (MD system “PT523”), with snapshots taken at 500 ns for n = 5 replicates. h, Snapshots from an MD simulation of docked PT523 sampled at various timepoints.
a, Select regions of a multiple sequence alignment of hSLC19A1, hSLC19A2, hSLC19A3. Numbering consistent with sequence position for hSLC19A1 b,The mutational effects of R133E and K411Q on the surface electrostatics of hRFCEM by APBS51 calculations in PyMOL. c, Ion probability densities from MD simulations of hRFCEM with and without in silico introduced mutants, becoming gradually more thiamine transporter-2 (hSLC19A3) like. Simulations performed in the presence of 150 mM KCl, with a threshold value of 25 shown for chloride or potassium for each simulation.
Extended Data Fig. 8 Antifolate drug recognition by human RFC and chicken proton-coupled folate transporter (PCFT).
a, Surface representation of the cavities for inward-facing hRFCEM-MTX and outward-facing pemetrexed (PMX)-bound PCFT. The orientation flip of the drugs in the cavities is highlighted by labelling the chemical groups. 0b, Chemical structure and immediate coordinating environment for MTX and PMX, with hydrogen bonds shown as red dashed lines and charged interactions highlighted by denoting charges. α- and γ- carboxylates are highlighted based on their extent of interactions in either system. c, The electrostatic environment of MTX and PMX as calculated by APBS61 of the hRFCEM-MTX and PCFT, respectively. Residues from b are shown as sticks with the anion binding site of RFC and selectivity pocket of PCFT as denoted.
This file contains Supplementary Figs. 1 and 2.
MD simulation of hRFC–MTX. A representative movie of a 1-µs MD simulation of hRFC–MTX, where MTX is covalently linked to K411. Functionally important residues E123, R133, R157 and R373 (green) and K411–MTX (salmon) are depicted as sticks. The protein in the cartoon is coloured by the N-terminal half (blue) and C-terminal half (yellow).
MD simulation of hRFC with unlinked MTX. A representative movie of a 2-µs MD simulation of hRFC with MTX unlinked from K411. MTX (salmon) and functionally important residues E123, R133, R157 and R373 (green) are shown as sticks. The protein in the cartoon is coloured by its N-terminal half (blue) and C-terminal half (yellow).
MD simulation of hRFC with PT523. A representative movie of a 0.7-µs MD simulation of hRFC with PT523 docked into the MTX–hRFC structure (with MTX removed). PT523 (salmon) and functionally important residues E123, R133, R157 and R373 (green) are shown as sticks. The protein in the cartoon is coloured by its N-terminal half (blue) and C-terminal half (yellow).
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Wright, N.J., Fedor, J.G., Zhang, H. et al. Methotrexate recognition by the human reduced folate carrier SLC19A1. Nature 609, 1056–1062 (2022). https://doi.org/10.1038/s41586-022-05168-0
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