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Recognition of cyclic dinucleotides and folates by human SLC19A1

Nature volume 612, pages 170–176 (2022)Cite this article

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Abstract

Cyclic dinucleotides (CDNs) are ubiquitous signalling molecules in all domains of life1,2. Mammalian cells produce one CDN, 2′3′-cGAMP, through cyclic GMP–AMP synthase after detecting cytosolic DNA signals3,4,5,6,7. 2′3′-cGAMP, as well as bacterial and synthetic CDN analogues, can act as second messengers to activate stimulator of interferon genes (STING) and elicit broad downstream responses8,9,10,11,12,13,14,15,16,17,18,19,20,21. Extracellular CDNs must traverse the cell membrane to activate STING, a process that is dependent on the solute carrier SLC19A122,23. Moreover, SLC19A1 represents the major transporter for folate nutrients and antifolate therapeutics24,25, thereby placing SLC19A1 as a key factor in multiple physiological and pathological processes. How SLC19A1 recognizes and transports CDNs, folate and antifolate is unclear. Here we report cryo-electron microscopy structures of human SLC19A1 (hSLC19A1) in a substrate-free state and in complexes with multiple CDNs from different sources, a predominant natural folate and a new-generation antifolate drug. The structural and mutagenesis results demonstrate that hSLC19A1 uses unique yet divergent mechanisms to recognize CDN- and folate-type substrates. Two CDN molecules bind within the hSLC19A1 cavity as a compact dual-molecule unit, whereas folate and antifolate bind as a monomer and occupy a distinct pocket of the cavity. Moreover, the structures enable accurate mapping and potential mechanistic interpretation of hSLC19A1 with loss-of-activity and disease-related mutations. Our research provides a framework for understanding the mechanism of SLC19-family transporters and is a foundation for the development of potential therapeutics.

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Fig. 1: Cryo-EM structure of apo hSLC19A1.
Fig. 2: Cryo-EM structures of hSLC19A1 in complexes with different CDNs.
Fig. 3: Interactions between hSLC19A1 and 2′3′-cGAMP.
Fig. 4: Cryo-EM structures of hSLC19A1 in complexes with 5-MTHF and PMX.
Fig. 5: The effects of SLC19A1 mutations and working models of SLC19A1-mediated substrate transport.

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Data availability

The cryo-EM maps have been deposited into the Electron Microscopy Data Bank under accession numbers EMD-33386 (apo hSLC19A1), EMD-33389 (hSLC19A1 + 2′3′-cGAMP), EMD-33387 (hSLC19A1 + 3′3′-CDA), EMD-33388 (hSLC19A1 + 2′3′-CDAS), EMD-34176 (hSLC19A1 + 5-MTHF) and EMD-34177 (hSLC19A1 + PMX). The coordinates have been deposited at the Protein Data Bank under accession numbers 7XPZ (apo hSLC19A1), 7XQ2 (hSLC19A1 + 2′3′-cGAMP), 7XQ0 (hSLC19A1 + 3′3′-CDA), 7XQ1 (hSLC19A1 + 2′3′-CDAS), 8GOE (hSLC19A1 + 5-MTHF) and 8GOF (hSLC19A1 + PMX).

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Acknowledgements

Cryo-EM data collection was carried out at the Center for Biological Imaging, Core Facilities for Protein Science at the Institute of Biophysics, Chinese Academy of Sciences. Computation work was performed using the high-performance computing resources at the Center for Biological Imaging, Institute of Biophysics, Chinese Academy of Science. All radioactivity experiments were performed at the Radioactive Isotope Laboratory (Institute of Biophysics, CAS), with guidance from H. J. Zhang in handling radioactive materials. We thank F. Wang and his group members for their help in antibody screening. The plasmid expressing membrane scaffold protein 1D1 (MSP1D1) was a gift from Z. Liu’s group. This work was supported by grants from National Key R&D Program of China (2018YFA0507203, 2018YFA0508000), National Natural Science Foundation of China (32130057, 31922037, 81921005, 32171219 and 82001681), Beijing Natural Science Foundation (Z220018), CAS Project for Young Scientists in Basic Research (YSBR-074), Strategic Priority Research Program of the Chinese Academy of Sciences (XDB37030203 and XDB29050100) and China Postdoctoral Science Foundation (BX2021039).

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Author notes

  1. These authors contributed equally: Qixiang Zhang, Xuyuan Zhang, Yalan Zhu, Panpan Sun

Authors and Affiliations

  1. CAS Key Laboratory of Infection and Immunity, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China

    Qixiang Zhang, Xuyuan Zhang, Panpan Sun, Liwei Zhang, Junxiao Ma, Lingan Zeng, Xiaohua Nie, Yina Gao, Zhaolong Li, Songqing Liu, Liguo Zhang & Pu Gao

  2. University of Chinese Academy of Sciences, Beijing, China

    Qixiang Zhang, Panpan Sun, Lingan Zeng, Zhaolong Li, Jizhong Lou, Liguo Zhang & Pu Gao

  3. School of Life Sciences, Beijing Institute of Technology, Beijing, China

    Yalan Zhu & Ang Gao

  4. Key Laboratory of RNA Biology, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China

    Yong Zhang & Jizhong Lou

  5. National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China

    Yina Gao, Songqing Liu & Pu Gao

  6. Science and Technology Innovation Center, Shandong First Medical University & Shandong Academy of Medical Sciences, Jinan, China

    Pu Gao

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  1. Qixiang Zhang
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Contributions

Q.Z., Y. Zhu and P.S. purified proteins, prepared cryo-EM samples, collected and processed cryo-EM data, and reconstructed density maps. Y. Zhu, Q.Z. and P.G. built and refined models. X.Z. and Q.Z. performed antibody screening and validation. X.Z. performed cellular assays. Y. Zhang, Z.L. and J.L. performed docking and molecular dynamics simulation. Liwei Zhang, J.M., X.N. and L. Zeng assisted with antibody screening. Y.G. assisted with cryo-EM data process and reconstruction. S.L. and A.G. assisted with cell culture and protein expression. P.G., Liguo Zhang and A.G. initiated the project and directed the research. P.G. wrote the manuscript with the help of all of the authors.

Corresponding authors

Correspondence to Ang Gao, Liguo Zhang or Pu Gao.

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Extended data figures and tables

Extended Data Fig. 1 Structure of hSLC19A1 and distribution of critical mutations.

a, Cut-open side view (left), side view (middle), and cytosolic bottom view (right) of hSLC19A1‘s conservation surface mapping using ConSurf. The cytosolic entrance is indicated by dashed white oval. b, Extracellular top view of the hSLC19A1 inward-open structure, with extracellular loops and TM1/2/7/8 helices highlighted in red. c, Hydrophobic interactions of IL6-7 (aa 204–214) (salmon) with surrounding elements (grey). Key residues are shown as sticks and dots. d, Hydrogen bonding interactions of IL6-7 (aa 204–214) with surrounding elements. Key residues are shown as sticks and hydrogen bonds are represented as dashed lines. e,f, Effect of IL6-7 (aa 204-214) mutant on extracellular CDN signalling. 293T-STING cells were transfected with empty vector, WT hSLC19A1, or IL6-7 (aa 204–214) mutant, and followed by 2′3′-cGAMP stimulation or left untreated. Immunoblot analyses were carried out using the antibodies of IRF3, phospho-IRF3, STING, α-Tubulin and SLC19A1. Representative results from four independent experiments are shown. Data are mean ± s.e.m. of n = 4 independent experiments. **, p ≤ 0.01 by unpaired, two-tailed Student‘s t-test. For gel sorce data, see Supplementary Fig. 3. g, Distribution of previously proposed important residues of hSLC19A1, as well as hSLC19A1 mutations found in antifolate drug resistant cell lines and cancer patients (from BioMuta database with functional prediction probability >0.99). For the corresponding human residue number, references and study strategies, see Supplementary Table 2.

Extended Data Fig. 2 Cryo-EM data processing of hSLC19A1 bound to 3′3′-CDA.

a, Representative raw micrograph of hSLC19A1-Fab-3′3′-CDA complex in nanodisc. b, Selected 2D class averages of hSLC19A1-Fab-3′3′-CDA complex particles. c, Data processing flow chart. d, Gold-standard Fourier shell correlation curves of the final reconstruction. e, Local resolution distribution of hSLC19A1-Fab-3′3′-CDA complex. f, Cryo-EM densities of transmembrane helices and intracellular loop IL6-7 at 5σ.

Extended Data Fig. 3 Cryo-EM data processing of hSLC19A1 bound to 2′3′-CDAS.

a, Representative raw micrograph of hSLC19A1-Fab-2′3′-CDAS complex in nanodisc. b, Selected 2D class averages of hSLC19A1-Fab-2′3′-CDAS complex particles. c, Data processing flow chart. d, Gold-standard Fourier shell correlation curves of the final reconstruction. e, Local resolution distribution of hSLC19A1-Fab-2′3′-CDAS complex. f, Cryo-EM densities of transmembrane helices and intracellular loop IL6-7 at 5σ.

Extended Data Fig. 4 Cryo-EM data processing of hSLC19A1 bound to 2′3′-cGAMP.

a, Representative raw micrograph of hSLC19A1-Fab-2′3′-cGAMP complex in nanodisc. b, Selected 2D class averages of hSLC19A1-Fab-2′3′-cGAMP complex particles. c, Data processing flow chart. d, Gold-standard Fourier shell correlation curves of the final reconstruction. e, Local resolution distribution of hSLC19A1-Fab-2′3′-cGAMP complex. f, Cryo-EM densities of transmembrane helices and intracellular loop IL6-7 at 5σ.

Extended Data Fig. 5 Chemical structures, densities, structural superimposition, and MD simulations of CDNs.

a–c, Chemical structures of 3′3′-CDA (a), 2′3′-CDAS (b), and 2′3′-cGAMP (c). d–f, Cryo-EM densities of 3′3′-CDA (d), 2′3′-CDAS (e), and 2′3′-cGAMP (f) at 8σ, 7σ, and 6σ, respectively. Two bound CDN molecules in all three structures are coloured as in Fig. 2. g, Structural superimposition for hSLC19A1 structures in apo and CDN-bound states. h, Cryo-EM densities of 3'3'-CDA at 2.5:1 (left; 7σ) and 0.5:1 (right; 5σ) ligand:protein molar ratios. i, The backbone RMSD of hSLC19A1 except F211-V250 from the cryo-EM structures for the simulation (top). The heavy atom RMSD of 3′3′-CDA from the initial position for the simulation (bottom). j–l, Alignments of initial state (violet) and final state (green) of the simulation for 3′3′-CDA-dimer (j), 3′3′-CDA-monomer-upper (k), and 3′3′-CDA-monomer-lower (l). m, Different conformations of the bound 3'3'-CDA, 2′3′-CDAS, and 2′3′-cGAMP based on structural superimposition results.

Extended Data Fig. 6 Interactions between hSLC19A1 and 3′3′-CDA/2′3′-CDAS.

a,c, Cut-open side view of electrostatic potential surface of hSLC19A1-3′3′-CDA (a) or hSLC19A1-2′3′-CDAS (c) complex. b,d, Slab through the surface of hSLC19A1 in a cytosolic bottom view, which highlights the binding position of 3′3′-CDA (b) or 2′3′-CDAS (d) relative to the cavity. e,f, Two views of stacking interactions between hSLC19A1 and 3′3′-CDA (e) or 2′3′-CDAS (f). g–j, Hydrogen bonding interactions between hSLC19A1 and 3′3′-CDA (g,h) or 2′3′-CDAS (i,j) from bottom/intracellular (g,i) and top/extracellular (h,j) views. All panels share similar representations and colour codes as in Fig. 3.

Extended Data Fig. 7 Effects of CDN-binding pocket mutants on extracellular CDN signalling.

a, Small conformational changes of the residues participated in CDN binding in 3′3′-CDA-, 2′3′-CDAS-, and 2′3′-cGAMP-bound structures. b,c, Effects of CDN-binding pocket mutants on extracellular CDN signalling. Experiments and quantification were performed as in Extended Data Fig. 1e, f. Data are mean ± s.e.m. of n = 3 independent experiments. *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001; NS, not significant. For gel sorce data, see Supplementary Fig. 3. d, Identification of SLC19A1 mutations in THP-1 and HeLa cell lines. The table (left) summaries the deletions and insertions for SLC19A1 alleles in THP-1 and HeLa cell lines. The diagrams (right) depict the predicted coding DNA sequence of SLC19A1 alleles in SLC19A1−/− cells. Boxes represent exons of SLC19A1 and horizontal lines represent introns. e, Expression of hSLC19A1 and its mutants in SLC19A1−/− THP-1 cells. Wild type and mutants were cloned into the lentiviral vector fusion with P2A-GFP which enables SLC19A1 and its mutant expression to be quantified via the expression of green fluorescence protein (GFP). SLC19A1 and its mutants (P2A-GFP+) were sorted and analysed by FACS. f,g Conserved function of SLC19A1-mediated CDN-transport. Comparison of CDN transport activities of human (h), mouse (m), and xenopus (x) SLC19A1. 293T-STING cells were transfected with SLC19A1 from different species, then the transfected cells were stimulated with 2′3′-cGAMP or left unstimulated. CDN transport activity was quantified by phospho-IRF3 and normalized to GFP (a surrogate for SLC19A1 expression). Data are mean ± s.e.m. of n=3 independent experiments. *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001 by two-tailed unpaired Student‘s t-test. For gel sorce data, see Supplementary Fig. 3.

Extended Data Fig. 8 Cryo-EM data processing of hSLC19A1 bound to 5-MTHF.

a, Representative raw micrograph of hSLC19A1-Fab-5-MTHF complex in nanodisc. b, Selected 2D class averages of hSLC19A1-Fab-5-MTHF complex particles. c, Data processing flow chart. d, Gold-standard Fourier shell correlation curves of the final reconstruction. e, Local resolution distribution of hSLC19A1-Fab-5-MTHF complex. f, Chemical structure of 5-MTHF. g, Cryo-EM densities of 5-MTHF at 8σ.

Extended Data Fig. 9 Cryo-EM data processing of hSLC19A1 bound to PMX.

a, Representative raw micrograph of hSLC19A1-Fab-PMX complex in nanodisc. b, Selected 2D class averages of hSLC19A1-Fab-PMX complex particles. c, Data processing flow chart. d, Gold-standard Fourier shell correlation curves of the final reconstruction. e, Local resolution distribution of hSLC19A1-Fab-PMX complex. f, Chemical structure of PMX. g, Cryo-EM densities of PMX at 3σ.

Extended Data Fig. 10 hSLC19A1-PMX interaction, protein expression, and different binding strategies for multiple substrates.

a, Hydrogen bonding interactions between PMX (sticks; wheat) and hSLC19A1 (grey). Key residues are shown as sticks and hydrogen bonds are represented as dashed lines. b, Hydrophobic interactions between PMX (sticks; wheat) and hSLC19A1 (grey). Key residues are shown as sticks and dots. c, Expression of hSLC19A1 and its mutants in SLC19A1−/− HeLa cells. Wild type and folate (left) or CDN (right) pocket mutants (P2A-GFP+) were sorted and quantified in HeLa cells by FACS. d–i, Structural superimposition between the pyrimidine-pyrrole moiety of PMX (sticks; wheat) and the adenine base of 3′3′-CDA (sticks; different conformations shown in different colours) was shown in the upper box. Slab through the surface of hSLC19A1 in a cytosolic bottom view (lower layer) highlights the steric hindrance effects formed between the modelled 3′3′-CDA (space-filling) and the cavity. The models of 3′3′-CDA in different conformations were obtained from: (d) PDB-4qsh, (e) the current work, (f) PDB-4qk9, (g) PDB-4qsh, and (h,i) predicted structures. j, Models of the CDN monomer docked into the cavity of hSLC19A1 using AutoDock Vina. k, Chemical structures of 5-MTHF (left), Folic acid (middle) and PT523 (right). l, Hydrophobic interactions between the methyl group on N5 of 5-MTHF (sphere; cyan) and Glu45, Ile48 and Tyr126 of hSLC19A1 (dots, grey). m, Hydrogen bond between N8 of 5-MTHF (sticks; cyan) and the carbonyl oxygen of Glu123 of hSLC19A1 (sticks; grey). n, Alignments of 50 ns structure snapshots from two independent MD simulations on hSLC19A1-PT523 models. o, Overall positively charged interior cavity (left) and cytosolic entrance (right) of hSCL19A1. NTD and CTD are coloured in yellow and blue, respectively; key residues are shown as sticks.

Extended Data Table 1 Cryo-EM data collection, refinement and validation statistics
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Zhang, Q., Zhang, X., Zhu, Y. et al. Recognition of cyclic dinucleotides and folates by human SLC19A1. Nature 612, 170–176 (2022). https://doi.org/10.1038/s41586-022-05452-z

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