Abstract
MFSD2A is a sodium-dependent lysophosphatidylcholine symporter that is responsible for the uptake of docosahexaenoic acid into the brain1,2, which is crucial for the development and performance of the brain3. Mutations that affect MFSD2A cause microcephaly syndromes4,5. The ability of MFSD2A to transport lipid is also a key mechanism that underlies its function as an inhibitor of transcytosis to regulate the blood–brain barrier6,7. Thus, MFSD2A represents an attractive target for modulating the permeability of the blood–brain barrier for drug delivery. Here we report the cryo-electron microscopy structure of mouse MFSD2A. Our structure defines the architecture of this important transporter, reveals its unique extracellular domain and uncovers its substrate-binding cavity. The structure—together with our functional studies and molecular dynamics simulations—identifies a conserved sodium-binding site, reveals a potential lipid entry pathway and helps to rationalize MFSD2A mutations that underlie microcephaly syndromes. These results shed light on the critical lipid transport function of MFSD2A and provide a framework to aid in the design of specific modulators for therapeutic purposes.
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Acknowledgements
We thank L. Montabana and D.-H. Chen for help with EM data collection. This work was made possible by support from Stanford University, the Harold and Leila Y. Mathers Charitable Foundation and NIA DP2AG052940 to L.F., a Dean’s fellowship to J.Z., the EMBO Long-Term Fellowship ALTF 544-2019 to D.A., an EMBO long-term fellowship to U.H.L., the NIH DP1 NS092473 Pioneer Award, the NIH/NINDS R35NS116820 grant, the Blavatnik Biomedical Accelerator grant and the QFASTR grant from Harvard Medical School to C.G. The research of C.G. was also supported in part by a Faculty Scholar grant from the Howard Hughes Medical Institute.
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C.A.P.W. and J.Z. carried out biochemical, functional and cryo-EM studies. D.A. carried out and analysed molecular dynamics simulations under the guidance of R.O.D. Y.X. assisted with functional and biochemical studies. B.A. and U.H.L. characterized the scFv. C.G. supervised the generation and characterizations of scFv. L.F. directed biochemical, functional and structural studies. C.A.P.W., J.Z. and L.F. wrote the manuscript with input from all authors.
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Extended data figures and tables
Extended Data Fig. 1 Sequence alignment of MFSD2A and MFSD2B homologues.
Sequence alignments of MFSD2A and MFSD2B from M. musculus (Mm), Homo sapiens (Hs), Danio rerio (Dr), Xenopus laevis (Xl), Bos taurus (Bt) and Gallus gallus (Gg) are shown.
Extended Data Fig. 2 Biochemical and functional characterizations of MFSD2A.
a, Size-exclusion chromatography profile of MFSD2A. b, Size-exclusion chromatography profile of MFSD2A in complex with scFv. c, Representative SDS–PAGE gel of purified MFSD2A–scFv complex. This was carried out four times in independent experiments. d, Uptake activity of Q67H used for structural studies. Uptake activity was normalized to that of the wild type (mean ± s.e.m., n = 4 biologically independent experiments). P values from one-way ANOVA followed by Tukey’s post hoc multiple comparison test are indicated on bar chart.
Extended Data Fig. 3 Single-particle cryo-EM analysis of MFSD2A.
a, Representative cryo-EM image of MFSD2A. b, Two-dimensional class averages of MFSD2A in CryoSparc. c, The workflow of classification and refinement. d, Angle distributions of the particles for the final reconstruction. e, Local resolution of the MFSD2A map calculated by MonoRes61. f, FSC of the final reconstruction as a function of resolution. Orange, gold-standard FSC curve between two half-maps from masked MFSD2A, with indicated resolution at FSC = 0.143; blue, FSC curve between the final atomic model and the local map masked on MFSD2A only, with indicated resolution at FSC = 0.5. FSC calculation performed by SAMUEL (SAM script)39.
Extended Data Fig. 4 Representative cryo-EM density maps of MFSD2A transmembrane helices.
Electron microscopy map density for 12 transmembrane helices of MFSD2A.
Extended Data Fig. 5 Conservation analysis of mouse MFSD2A structure.
Residues are coloured from variable to conserved according to the palette below the structure.
Extended Data Fig. 6 Intracellular elements of MFSD2A.
Ribbon representation (left) and cylindrical representation (right) of MFSD2A viewed from the intracellular side. N- and C-domains are coloured in cyan and green, respectively. IL, intracellular linker (orange). The helix after the last transmembrane helix is also coloured in orange.
Extended Data Fig. 7 Sodium-binding sites in molecular dynamics simulations.
a, Shaded regions (black, blue and green) indicate points in time during each simulation when a sodium ion was present at the Na1 site—in particular, points at which a sodium ion at a distance of 2–5 Å from the T95 side-chain oxygen formed a salt bridge with D92 and/or D96. b, Shaded regions indicate points in time during each simulation when a sodium ion was present at the Na2 site—in particular, points at which a sodium ion at a distance of 5–8 Å from the T95 side-chain oxygen formed a salt bridge with D92 and/or E159. Both a and b show data for simulations under three conditions. In the first two conditions (black and blue), a sodium ion is initially placed in the binding pocket at a position suggested by the potential coordination environment and the cryo-EM density, whereas in the third (black), no sodium ions are initially placed in the binding pocket. The first and third conditions (black and green) used a 9 Å nonbonded interaction cut-off, whereas the second (blue) used a 12 Å cut-off. Plots include equilibration as well as production phases of each simulation. c, Sodium positions from simulation no. 2 of the first condition (highlighted by red box), in which sodium ions bind simultaneously at the Na1 and Na2 sites. Positions of sodium bound at the Na1 site are shown as purple spheres, and positions of sodium bound at the Na2 site are shown as orange spheres (Methods). In this simulation, a Na+ ion was initially placed at a position proposed on the basis of the potential coordination environment and the cryo-EM density map, shown as a yellow circle. d, Sodium-binding sites in a representative frame from the same simulation. Sodium-coordinating residues are shown as sticks. Sodium bound at the Na1 site is shown as a purple sphere and sodium bound at the Na2 site is shown as an orange sphere. Oxygen atoms of water molecules are shown as red spheres.
Extended Data Fig. 8 Structural mapping of disease-causing mutations.
a, Close-up view of S170. S170 and R190 (sticks) are within hydrogen-bond distance. b, Zoomed-in view of S343, near the helical bend of TM8 that gives rise to lateral opening. c, Uptake activities of mouse MFSD2A variants with equivalent point mutations to human microcephaly-associated mutations. Uptake activities are normalized to that of the wild type (mean ± s.e.m., n = 6 biologically independent experiments). P values from one-way ANOVA followed by Tukey’s post hoc multiple comparison test are indicated on bar chart.
Extended Data Fig. 9 Structure of MFSD2A in complex with scFv.
a, The cryo-EM map of the MFSD2A–scFv complex. b, The ribbon representations of the MFSD2A–scFv complex. A model scFv was docked into the density.
Supplementary information
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This file contains an example of the gating strategy for flow cytometry in uptake assays.
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Wood, C.A.P., Zhang, J., Aydin, D. et al. Structure and mechanism of blood–brain-barrier lipid transporter MFSD2A. Nature 596, 444–448 (2021). https://doi.org/10.1038/s41586-021-03782-y
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DOI: https://doi.org/10.1038/s41586-021-03782-y
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