Molecular basis of sphingosine-1-phosphate transport by MFSD2B provides insights for selective inhibitor screening
Abstract
Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid that regulates immune cell trafficking, vascular homeostasis, and platelet function. MFSD2B, a major facilitator superfamily transporter, maintains plasma S1P levels by exporting S1P from erythrocytes and platelets, yet the molecular basis of S1P recognition and transport by MFSD2B remains unclear. Here, we report a 3.65 Å cryo-EM structure of Sus scrofa MFSD2B captured in an outward-facing conformation. Together with AlphaFold3-based modeling, molecular docking and dynamics simulations, and mutagenesis-based transport assays, our results identify a distributed S1P-recognition pathway required for MFSD2B transport. Comparative analyses show that MFSD2B preserves the lipid-transport features of MFSD2A but lacks LPC-recognition and Na⁺-coupling sites. Despite sharing S1P transport activity, MFSD2B and SPNS2 recognize the phosphate headgroup of S1P through distinct pocket architectures. Current S1P-mimic SPNS2 inhibitors cross-react with MFSD2B, highlighting the need for scaffold-divergent inhibitors with improved transporter selectivity. We establish a transporter-receptor conjugated screening (TRCS) system to enable efficient and cost-effective inhibitor screening. Together, our findings provide a structural framework for S1P transport by MFSD2B and may facilitate the discovery of selective lipid transporter inhibitors.
References
1. Ren., R., et al., A Glimpse of the Structural Biology of the Metabolism of Sphingosine-1-Phosphate. Contact: The Journal of Inter-Organelle Communication, 2021. 4(2021).
2. Pappu, R., et al., Promotion of Lymphocyte Egress into Blood and Lymph by Distinct Sources of Sphingosine-1-Phosphate. Science, 2007. 316(5822): p. 295–298.
3. Kawahara, A., et al., The Sphingolipid Transporter Spns2 Functions in Migration of Zebrafish Myocardial Precursors. Science, 2009. 323(5913): p. 524–527.
4. Vu, T.M., et al., Mfsd2b is essential for the sphingosine-1-phosphate export in erythrocytes and platelets. Nature, 2017. 550(7677): p. 524–528.
5. Fukuhara, S., et al., The sphingosine-1-phosphate transporter Spns2 expressed on endothelial cells regulates lymphocyte trafficking in mice. The Journal of Clinical Investigation, 2012. 122(4): p. 1416–1426.
6. Ghaderi, S. and B. Levkau, An erythrocyte-centric view on the MFSD2B sphingosine-1-phosphate transporter. Pharmacology & Therapeutics, 2023. 249: p. 108483.
7. Yan, N., Structural Biology of the Major Facilitator Superfamily Transporters. Annual Review of Biophysics, 2015. 44(Volume 44, 2015): p. 257–283.
8. Nguyen, L.N., et al., Mfsd2a is a transporter for the essential omega-3 fatty acid docosahexaenoic acid. Nature, 2014. 509(7501): p. 503–506.
9. Nguyen, C., et al., Lipid flipping in the omega-3 fatty-acid transporter. Nature Communications, 2023. 14(1): p. 2571.
10. Pang, B., et al., Molecular basis of Spns2-facilitated sphingosine-1-phosphate transport. Cell Research, 2024. 34(2): p. 173–176.
11. Wood, C.A.P., et al., Structure and mechanism of blood–brain-barrier lipid transporter MFSD2A. Nature, 2021. 596(7872): p. 444–448.
12. Chen, H., et al., Structural and functional insights into Spns2-mediated transport of sphingosine-1-phosphate. Cell, 2023. 186(12): p. 2644–2655.e16.
13. Dastvan, R., et al., Proton-driven alternating access in a spinster lipid transporter. Nature Communications, 2022. 13(1): p. 5161.
14. Donoviel, M.S., et al., Spinster 2, a sphingosine-1-phosphate transporter, plays a critical role in inflammatory and autoimmune diseases. The FASEB Journal, 2015. 29(12): p. 5018–5028.
15. Tanaka, S., et al., Sphingosine 1-phosphate signaling in perivascular cells enhances inflammation and fibrosis in the kidney. Science Translational Medicine, 2022. 14(658): p. eabj2681.
16. Foster, D.J., et al., Discovery of Potent, Orally Bioavailable Sphingosine-1-Phosphate Transporter (Spns2) Inhibitors. Journal of Medicinal Chemistry, 2024. 67(13): p. 11273–11295.
17. Wu, X., et al., An LC-MS-based workflow measures the export activity of S1P transporters. Biochemical and Biophysical Research Communications, 2023. 668: p. 118–124.
18. Yu, L., et al., Structural insights into subtype-specific agonist recognition by sphingosine-1-phosphate receptors. PLOS Biology, 2026. 24(4): p. e3003381.
19. Mastronarde, D.N., SerialEM: A Program for Automated Tilt Series Acquisition on Tecnai Microscopes Using Prediction of Specimen Position. Microscopy and Microanalysis, 2003. 9(S02): p. 1182–1183.
20. Zheng, S.Q., et al., MotionCor2: anisotropic correction of beam-induced motion for improved cryo-electron microscopy. Nature Methods, 2017. 14(4): p. 331–332.
21. Punjani, A., et al., cryoSPARC: algorithms for rapid unsupervised cryo-EM structure determination. Nature Methods, 2017. 14(3): p. 290–296.
22. Zhu, J., et al., A minority of final stacks yields superior amplitude in single-particle cryo-EM. Nature Communications, 2023. 14(1): p. 7822.
23. Bienert, S., et al., The SWISS-MODEL Repository—new features and functionality. Nucleic Acids Research, 2017. 45(D1): p. D313–D319.
24. Emsley, P. and K. Cowtan, Coot: model-building tools for molecular graphics. Acta Crystallographica Section D, 2004. 60(12 Part 1): p. 2126–2132.
25. Adams, P.D., et al., PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallographica Section D, 2010. 66(2): p. 213–221.
26. Amunts, A., et al., Structure of the Yeast Mitochondrial Large Ribosomal Subunit. Science, 2014. 343(6178): p. 1485–1489.
27. Chen, V.B., et al., MolProbity: all-atom structure validation for macromolecular crystallography. Acta Crystallographica Section D, 2010. 66(1): p. 12–21.
28. Pettersen, E.F., et al., UCSF Chimera—A visualization system for exploratory research and analysis. Journal of Computational Chemistry, 2004. 25(13): p. 1605–1612.
29. Pettersen, E.F., et al., UCSF ChimeraX: Structure visualization for researchers, educators, and developers. Protein Science, 2021. 30(1): p. 70–82.
30. Eberhardt, J., et al., AutoDock Vina 1.2.0: New Docking Methods, Expanded Force Field, and Python Bindings. Journal of Chemical Information and Modeling, 2021. 61(8): p. 3891–3898.
31. O'Boyle, N.M., et al., Open Babel: An open chemical toolbox. Journal of Cheminformatics, 2011. 3(1): p. 33.
32. Abramson, J., et al., Accurate structure prediction of biomolecular interactions with AlphaFold 3. Nature, 2024. 630(8016): p. 493–500.
33. Jo, S., et al., CHARMM-GUI: A web-based graphical user interface for CHARMM. Journal of Computational Chemistry, 2008. 29(11): p. 1859–1865.
34. Lomize, M.A., et al., OPM database and PPM web server: resources for positioning of proteins in membranes. Nucleic Acids Res, 2012. 40(Database issue): p. D370–6.
35. Huang, J. and A.D. MacKerell Jr, CHARMM36 all-atom additive protein force field: Validation based on comparison to NMR data. Journal of Computational Chemistry, 2013. 34(25): p. 2135–2145.
36. Abraham, M.J., et al., GROMACS: High performance molecular simulations through multi-level parallelism from laptops to supercomputers. SoftwareX, 2015. 1-2: p. 19–25.
37. Berendsen, H.J.C., et al., Molecular dynamics with coupling to an external bath. The Journal of Chemical Physics, 1984. 81(8): p. 3684–3690.
38. Parrinello, M. and A. Rahman, Crystal Structure and Pair Potentials: A Molecular-Dynamics Study. Physical Review Letters, 1980. 45(14): p. 1196–1199.
39. Bussi, G., D. Donadio, and M. Parrinello, Canonical sampling through velocity rescaling. The Journal of Chemical Physics, 2007. 126(1).
40. Darden, T., D. York, and L. Pedersen, Particle mesh Ewald: An N⋅log(N) method for Ewald sums in large systems. The Journal of Chemical Physics, 1993. 98(12): p. 10089–10092.
41. Applegate DL, B.R., Chvátal V, & Cook WJ, The Traveling Salesman Problem, in Combinatorial Optimization. 1997. p. 241–271.
Metrics
DOI:
Submission ID:
Downloads
Posted
Versions
- 2026-05-19 (2)
- 2026-05-18 (1)
How to Cite
Download Citation
Declaration of Competing Interests
The authors declare no competing interests to disclose.
Copyright
The copyright holder for this preprint is the author/funder.
This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.