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Structural basis of omega-3 fatty acid transport across the blood–brain barrier

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Abstract

Docosahexaenoic acid is an omega-3 fatty acid that is essential for neurological development and function, and it is supplied to the brain and eyes predominantly from dietary sources1,2,3,4,5,6. This nutrient is transported across the blood–brain and blood–retina barriers in the form of lysophosphatidylcholine by major facilitator superfamily domain containing 2A (MFSD2A) in a Na+-dependent manner7,8. Here we present the structure of MFSD2A determined using single-particle cryo-electron microscopy, which reveals twelve transmembrane helices that are separated into two pseudosymmetric domains. The transporter is in an inward-facing conformation and features a large amphipathic cavity that contains the Na+-binding site and a bound lysolipid substrate, which we confirmed using native mass spectrometry. Together with our functional analyses and molecular dynamics simulations, this structure reveals details of how MFSD2A interacts with substrates and how Na+-dependent conformational changes allow for the release of these substrates into the membrane through a lateral gate. Our work provides insights into the molecular mechanism by which this atypical major facility superfamily transporter mediates the uptake of lysolipids into the brain, and has the potential to aid in the delivery of neurotherapeutic agents.

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Fig. 1: Structure of MFSD2A in an inward-facing conformation.
Fig. 2: The intracellular cavity of MFSD2A features the Na+- and lysolipid-binding sites.
Fig. 3: Molecular dynamics simulations of MFSD2A reveal coupling between Na+ binding and lysolipid movement through a dynamic intracellular gate.
Fig. 4: Proposed mechanism of MFSD2A-mediated transport.

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Data availability

All raw movie frames have been deposited into EMPIAR, with accession code EMPIAR-10698. The density map has been deposited into Electron Microscopy Data Bank, with accession code EMD-23883. The model has been deposited in the PDB, with accession code 7MJS. All raw flow cytometry data and gels are available in the Article or its Supplementary Information. Any additional data are available from the corresponding authors upon reasonable request.

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Acknowledgements

We thank members of the laboratory of F.M., Columbia Cryo-EM facility and Iowa State University Protein Facility for their assistance; and E. Kots for generating the molecular dynamics trajectory video. This work was supported by NIH grants (R35 GM132120 and R21 MH125649 to F.M., R35 GM128624 to M.T.M. and R01 GM117372 to A.A.K.) and grants from the National Research Foundation and Ministry of Health, Singapore (NRF-NRFI2017-05 and MOH-000217 to D.L.S.). R.J.C. was supported by the Simons Society of Fellows (award number 578646). G.K. is supported by the HRH Prince Alwaleed Bin Talal Bin Abdulaziz Alsaud Institute of Computational Biomedicine at Weill Cornell Medical College through the 1923 Fund. C.F.C. and B.H.W. are supported by the Khoo Postdoctoral Research Fellowship. Some of the work was performed at the Center for Membrane Protein Production and Analysis (COMPPÅ; NIH P41 GM116799 to W. A. Hendrickson), and at the National Resource for Automated Molecular Microscopy at the Simons Electron Microscopy Center (P41 GM103310), both located at the New York Structural Biology Center. Molecular dynamics simulations were performed using the Oak Ridge Leadership Computing Facility (summit allocation BIP109) at the Oak Ridge National Laboratory (supported by the Office of Science of the US Department of Energy under contract number DE-AC05-00OR22725), and computational resources of the David A. Cofrin Center for Biomedical Information at Weill Cornell Medical College.

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Authors and Affiliations

  1. Department of Physiology and Cellular Biophysics, Columbia University, New York, NY, USA

    Rosemary J. Cater, Brendon C. Choy, Joseph G. Pepe, Giacomo Parisi, Oliver B. Clarke & Filippo Mancia

  2. Signature Research Program in Cardiovascular and Metabolic Disorders, Duke-NUS Medical School, Singapore, Singapore

    Geok Lin Chua, Cheen Fei Chin, Debra Q. Y. Quek, Bernice H. Wong & David L. Silver

  3. Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, IL, USA

    Satchal K. Erramilli, Piotr Tokarz & Anthony A. Kossiakoff

  4. Department of Chemistry and Biochemistry, University of Arizona, Tucson, AZ, USA

    James E. Keener & Michael T. Marty

  5. Center on Membrane Protein Production and Analysis, New York Structural Biology Center, New York, NY, USA

    Brian Kloss

  6. Department of Anesthesiology, Columbia University Irving Medical Center, New York, NY, USA

    Oliver B. Clarke

  7. Department of Physiology and Biophysics, Weill Cornell Medical College, Cornell University, New York, NY, USA

    George Khelashvili

  8. Institute for Computational Biomedicine, Weill Cornell Medical College, Cornell University, New York, NY, USA

    George Khelashvili

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  1. Rosemary J. Cater
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Contributions

R.J.C. performed cloning with assistance from B.K., designed and performed expression screening experiments, produced baculovirus, and optimized protein expression and purification with assistance from B.C.C. Fabs were identified and purified by S.K.E., P.T. and A.A.K.  Protein preparation for structural analysis was performed by R.J.C. who also screened and optimized sample vitrification, and generated cryo-EM data with assistance from G.P. Cryo-EM data analysis and model building was performed by R.J.C. with guidance from O.B.C. Uptake studies and mutagenesis experiments were designed and performed by G.L.C., C.F.C., D.Q.Y.Q. and D.L.S. Molecular dynamics simulations were designed and performed by G.K., with input from R.J.C. and F.M. TLC analysis was performed by G.L.C. and B.H.W. Samples for native mass spectrometry were prepared by R.J.C. and J.G.P. Native mass spectrometry was performed by J.E.K. and M.T.M. The manuscript was written by R.J.C., F.M. and G.K. with input from G.L.C. and D.L.S. Figures were prepared by R.J.C., G.K., B.C.C. and G.L.C. Guidance and input at all stages of the project were provided by F.M. and D.L.S.

Corresponding authors

Correspondence to George Khelashvili, David L. Silver or Filippo Mancia.

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Competing interests

D.L.S. is a scientific founder and advisor of Travecta Therapeutics that has developed a drug delivery platform that uses MFSD2A transport. All other authors declare no competing interests.

Additional information

Peer review information Nature thanks Osamu Nureki, Diwakar Shukla and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data figures and tables

Extended Data Fig. 1 Biochemical characterization, functional validation, nanodisc reconstitution and Fab complex formation of GgMFSD2A.

a, Chemical structure of LPC-DHA. b, Fluorescent size-exclusion chromatography elution profiles of seven MFSD2A orthologues fused to green fluorescent protein. Orthologues screened are from D. rerio (MFSD2A_DR) (NCBI BC085388), X. tropicalis (MFSD2A_XT) (NCBI BC123088), B. taurus (MFSD2A_BT) (NCBI BC149727), GgMFSD2A (MFSD2A_GG) (NCBI XM_417826), C. lupus familiaris (MFSD2A_CLF) (NCBI XP_532546), HsMFSD2A (MFSD2A_HS) (NCBI NM_032793) and M. musculus (MFSD2A_MM) (NCBI NM_029662). c, MFSD2A-mediated uptake of C14-LPC-18:1 into HEK293 cells transfected with either HsMFSD2A or GgMFSD2A wild-type and Na+-binding-deficient mutant constructs (D97A and D92A, respectively). Uptake is expressed as mean ± s.e.m.; n = 3 independent experiments. d, Complementarity-determining region (CDR) sequences of unique synthetic antigen binders (Fabs) panned for binding to iGgMFSD2A reconstituted in MSP1E3D1 nanodiscs. Enriched Tyr-Ser-Gly-Trp residues, which have an important role in antigen recognition, are highlighted in purple, blue, red and orange, respectively; other conserved residues are highlighted in green. Residues are numbered according to the Kabat system78. e, EC50 evaluation of purified Fabs 2AG1 (blue) (1.71 ± 0.03 nM), 2AG2 (red) (2.06 ± 0.08 nM), and 2AG3 (orange) (8.94 ± 0.08 nM) binding to GgMFSD2A incorporated into MSP1E3D1 nanodiscs. Data points represent mean ± s.d.; n = 3 independent experiments. f, Normalized high-performance liquid chromatography elution profiles of GgMFSD2A purified in DDM supplemented with CHS (black) and reconstituted in MSP1E3D1 nanodiscs without (blue) and with (green) 2AG3 Fab bound. g, Representative SDS–PAGE gel of purified GgMFSD2A reconstituted in MSP1E3D1 nanodiscs with 2AG3 Fab bound. For gel source data, see Supplementary Fig. 1.

Extended Data Fig. 2 Multiple sequence alignment of MFSD2A and MFSD2B.

Seven MFSD2A and six MFSD2B variants were aligned using MUSCLE57 and visualized and coloured using Jalview with the ClustalX colour scheme79. The sequences aligned for MFSD2A are from D. rerio (NCBI BC085388) (2A_DR), X. tropicalis (NCBI BC123088) (2A_XT), B. taurus (NCBI BC149727) (2A_BT), G. gallus (NCBI XM_417826) (2A_GG), C. lupus familiaris (NCBI XP_532546) (2A_CLF), H. sapiens (NCBI-NM_032793) (2A_HS) and M. musculus (NCBI-NM_029662) (2A_MM). The sequences aligned for MFSD2B are from X. tropicalis (UniProtKB A4IH46) (2B_XT), B. taurus (NCBI XM_010810291) (2B_BT), G. gallus (NCBI- XM_004935790) (2B_GG), C. lupus familiaris (NCBI XM_005630178) (2B_CLF), H. sapiens (NCBI NM_001346880) (2B_HS) and M. musculus (NCBI NM_001033488) (2B_MM). Secondary structural elements are shown as cylinders, labelled and coloured as in Fig. 1. Residues discussed throughout the Article are indicated with circles of the same colour used to highlight them in figure panels showing the structure. Additionally, red diamonds denote known disease-causing human mutations12,14,15,16, green hexagons denote glycosylation sites (in GgMFSD2A) and yellow triangles denote two cysteines that form a disulfide crosslink.

Extended Data Fig. 3 Cryo-EM workflow and analysis of the MFSD2A–Fab 2AG3 complex.

a, Flow chart outlining cryo-EM image acquisition and processing performed to obtain a structure of nanodisc-reconstituted GgMFSD2A in complex with the Fab 2AG3. A representative micrograph and 2D class averages are shown. Although 2D class averages of monomeric and dimeric GgMFSD2A with and without bound Fab were observed, only monomeric particles with bound Fab (green) were used for the final reconstruction; the others (red) were discarded. All processing was performed using CryoSPARC v.2.1543 (Methods). b, Euler angle distribution plot of the final three-dimensional reconstruction of the GgMFSD2A–Fab 2AG3 complex. c, Fourier shell correlation (FSC) curves for the GgMFSD2A–Fab 2AG3 complex. d, Local resolution map of the GgMFSD2A–Fab 2AG3 complex, with an orthogonal view indicating the location of the clipping plane.

Extended Data Fig. 4 Fit of cryo-EM density with MFSD2A model.

Cryo-EM densities (semi-transparent surface) are superimposed on structural elements of GgMFSD2A, including TM1 to TM12, C207–C460 disulfide crosslink, N-linked glycans and bound LPC-18:3 in two possible conformations. TMs are rendered as a cartoon with residue side chains in stick representation and coloured as in Fig. 1; the other features are also shown in stick representation.

Extended Data Fig. 5 Characterization of an endogenous lysolipid bound to MFSD2A.

a, Native (left) and zero-charge deconvolved (right) mass spectra of GgMFSD2A in DDM. Peaks corresponding to glycosylated GgMFSD2A with no bound ligand are highlighted in orange, whereas peaks corresponding to glycosylated GgMFSD2A with a bound lysolipid (515 Da) are highlighted in purple. b, Single-cell uptake of the fluorescent substrate LPC–NBD by cells expressing wild-type and D97A (corresponding to D92 in GgMFSD2A) HsMFSD2A–mCherry fusion constructs analysed by fluorescence-activated cell sorting. LPC–NBD uptake was measured in the presence of increasing concentrations of LPC-18:3 (the number of cells analysed per condition was around 900). Left panels show output from flow cytometric analysis (Methods, Extended Data Fig. 6), and right panels show gated data normalized to mCherry expression and represented as a per cent of uptake by wild type in the absence of LPC-18:3 (denoted as WTmax on axis), collected in the same experiment ± s.e.m. c, TLC analysis of cells expressing wild-type and D97A HsMFSD2A showing intracellular conversion of LPC-18:3 to PC-18:3 as evidence for LPC-18:3 uptake7. Experiments were performed in triplicate and the quantified intensities of the PC bands are indicated for each lane. d, Native (left) and zero-charge deconvolved (right) mass spectra of GgMFSD2A in POPG-filled MSP1E3D1 nanodiscs. Apo and lysolipid-bound peaks are coloured as in a, and those annotated with an asterisk correspond to an unassigned mass of 68,980 Da, which may be attributed to a glycan cleavage or truncation of the protein.

Extended Data Fig. 6 Flow cytometry analysis and concentration response for HsMFSD2A-mediated uptake of LPC–NBD.

a, Top panels, single-cell events were separated from doublets for wild-type and D97A HsMFSD2A–mCherry constructs via selection of populations through the area (A), width (W) and height (H) of the mCherry fluorescence intensities for forward (FSC) and sideward (SSC) scatter, performed in sequence as indicated by black arrows. Middle panels, selected single cells (indicated by ovals and rectangles at each stage in the top panels) were analysed for LPC–NBD and mCherry fluorescence, as well as viability. LPC–NBD fluorescence intensity is represented as a rainbow colour gradient from blue (low) to red (high). Viability was assessed by DAPI staining, and non-viable cells are annotated as ‘population d’ and number of non-viable cells is indicated in the top left corner of each respective plot. Gates for subsequent analyses were set as described in the Methods and are indicated by a black rectangle. The number of cells within each gate is indicated in the top left corner of each respective plot. Bottom, scatter plot of single-cell uptake of LPC–NBD by gated cells expressing wild-type (black) and D97A (red) HsMFSD2A–mCherry fusion constructs. LPC–NBD uptake was normalized to mCherry expression and represented as a per cent of wild type ± s.e.m. Horizontal bar indicates the mean, and each point represents a single-cell event. The number of cells plotted is indicated in the top left corner of the respective plot in the middle panel. b, Top, uptake of increasing concentrations of LPC–NBD by single cells transfected with either wild-type or D97A HsMFSD2A–mCherry constructs. Data were analysed as in a, and the number of cells analysed per condition was about 1,000. Bottom, LPC–NBD concentration response curves for wild-type (black) and D97A (red) HsMFSD2A–mCherry fusion constructs. Hill coefficient (n) and concentration for half-maximal LPC–NBD uptake (EC50) for wild type are indicated on the graph. c, Flow cytometric analysis of LPC–NBD and mCherry fluorescence for wild-type and all analysed mutant HsMFSD2A constructs. LPC–NBD fluorescence intensity is represented as a rainbow colour gradient from blue (low) to red (high). Gates and indicated by a black rectangle and the number of cells within each gate is shown in the top left corner of each plot. d, Scatter plot of single-cell uptake of LPC–NBD by gated cells expressing wild-type and mutant HsMFSD2A–mCherry fusion constructs. LPC–NBD uptake was normalized to mCherry expression and represented as a per cent of wild type ± s.e.m. Horizontal bar indicates the mean, and each point represents a single cell event. The n values for each construct assayed are in c. The colour coding matches that used in Fig. 2, Extended Data Fig. 11. In cases in which residue number or identity differ between HsMFSD2A and GgMFSD2A, the corresponding residue in GgMFSD2A is provided in parentheses.

Extended Data Fig. 7 tICA analysis identifies major conformational states sampled in molecular dynamics simulations and reveals their structural characteristics.

a, Two-dimensional (2D) landscape representing all molecular dynamics trajectories mapped with the tICA transformation in the space of the first two tICA eigenvectors (tIC1 and tIC2). Population distribution within the 2D space is indicated by a colour gradient with the red and blue shades representing the most and least populated regions of 2D space, respectively. Locations of 50 microstates obtained from k-means clustering analysis of the tICA space are shown as small black squares. b, Contribution of each tICA vector to the total fluctuation of the system. c, Contributions of the collective variables used as tICA parameters to tIC1 and tIC2. d, The 2D tICA space from a, highlighting 12 selected microstates (labelled 1 to 12) that describe structural properties of various conformational states of the system. The three shaded regions represent conformations of the protein with no ion bound (green shade), ion bound at D92 (red shade) and ion bound at E312 (blue shade). e, The 2D tICA space from a, redrawn to show the direction of change in the extent of lysolipid penetration and intracellular gate (IC-gate) opening along the landscape. f, Columns from left to right show probability distributions of the following collective variables in the 12 microstates selected from d: vertical distance (Z) between lysolipid phosphorous atom (Plyso) and E312 Cα, number of lysolipid atoms in the intracellular cavity, minimum distances between any ion in the system and D92, between any ion in the system and E312, between M182 and W403, between M182 and F399, between R85 and D88, between R85 and E312, and number of water oxygen atoms in the intracellular cavity. The ranges of collective variables were normalized such that the lowest and highest values for each variable correspond to 0 and 1, respectively. State numbers are provided on the lefthand side as (i)–(iv), which correspond to Fig. 3a–d, respectively.

Extended Data Fig. 8 Lipid penetration into the intracellular cavity of MFSD2A.

a, Snapshots depicting the position of phosphorus atoms (spheres) of POPC (orange), LPC-18:1 (purple), LPC-18:3 (teal) and LPC-DHA (lime) at the entrance to and inside the intracellular cavity during molecular dynamics simulations. b, Histograms representing the distance along the membrane-perpendicular z axis between the phosphorus atoms of the lipids in a, and the Cα atom of a reference residue (E312, highlighted in a,). c, Structure of LPC-18:3 highlighting the headgroup (green; O11, O12, O13, O14, P, C11, C12, N, C13, C14 and C15 in CHARMM nomenclature), backbone (blue; C31, O31, O32, C3, C2, O21 and C1), single-bonded hydrocarbon tail (pink; C32–C38), and double-bonded hydrocarbon tail (yellow; C39–C48) regions. d, Frequency of contacts between the LPC-18:3 headgroup, backbone, single-bonded and double-bonded tail regions, and GgMFSD2A residues. Results for top 20 contact residues are shown. Contacts were defined as distances less than 4 Å between non-hydrogen atoms of the protein and the substrate.

Extended Data Fig. 9 Sampling of the major conformational states is affected by the presence and absence of Na+ and lysolipid substrate in the molecular dynamics simulations.

Projection (coloured dots) of each molecular dynamics trajectory from the three sets of simulations on the 2D tICA landscape (in pale colours) from Extended Data Fig. 7. a–d, Simulations of GgMFSD2A in a POPC bilayer were performed in either Na+Cl− or Li+Cl− solution (a); Na+Cl− solution with a single LPC-18:1 at the interface of the intracellular gate and the inner leaflet of the membrane (b); Na+Cl− solution with a single LPC-18:3 at the interface of the intracellular gate and the inner leaflet of the membrane (c); and Na+Cl− solution with a single LPC-18:3 bound as in the cryo-EM structure (d). The colours of the dots indicate the timeframes in the evolution of the trajectory: blue and cyan represent the initial stages of the simulation, yellow and green correspond to the middle part of the trajectory, and red shows the last third of the trajectory. The yellow circle in a (trajectory 0) denotes the conformation of the system in which LPC-18:1, LPC-18:3 and LPC-DHA were introduced.

Extended Data Fig. 10 Pathways for opening of the intracellular gate and substrate entry.

a, The three main pathways for intracellular gate (IC-gate) opening and substrate penetration, identified from Markov state modelling and transition path theory analyses are shown on the tIC1 versus tIC2 landscape, represented as in Extended Data Fig. 7. We named the macrostates on these pathways A to H; each label is placed at the centre of the corresponding state. Arrows coloured in blue, red and green represent the three main pathways between macrostates, and their thickness indicates the relative magnitude of flux for each given pathway. Macrostates A, I, G and H correspond to states shown in Fig. 3a, 3b, 3c and 3d, respectively, and macrostate E corresponds to the cryo-EM structure. The remaining macrostates represent structural intermediates between these major states. b, The percentage of total flux for the top three pathways shown in a. c, Structural representation of the key macrostates from a. Macrostates B, C, F and K have been omitted for simplicity, as they are structural intermediates between the major states presented (Extended Data Fig. 7). Helices in the N domain are in yellow; helices in the C domain are in green. Select residues are shown in stick representation and coloured as in Fig. 2. For visual clarity, TM1, TM3, TM4, TM6, TM9 and TM12 have been omitted, and TM5 is partially transparent.

Extended Data Fig. 11 Interdomain contacts on the extracellular side of MFSD2A.

a, Structure of GgMFSD2A in the plane of the membrane with inset highlighting a disulfide crosslink between C207 and C460 (salmon), a charged and polar network involving D68, Y321, R326 and Y455 (brown), and a hydrophobic plug comprising F60, F61, F329 and L333 (grey). TMs are coloured as in Fig. 1. b, Single-cell uptake of the fluorescent substrate LPC–NBD by cells expressing wild-type and mutant HsMFSD2A–mCherry fusion constructs. LPC–NBD uptake was normalized to mCherry expression and represented as a per cent of wild type collected in the same experiment ± s.e.m. The colour coding matches that used in a. In cases in which residue number or identity differs between HsMFSD2A and GgMFSD2A the corresponding residue in GgMFSD2A is provided in parentheses. c, d, Snapshots from molecular dynamics simulations of wild-type (c) and F60A/F61A/F329A/L333A-mutant GgMFSD2A (d), showing water oxygens as purple spheres. Insets illustrate magnified views of the intracellular cavity. GgMFSD2A is represented as in Fig. 1, with plug residues labelled and coloured in grey. e, Number of water oxygens in the intracellular cavity derived from simulations of wild-type and F60A/F61A/F329A/L333A-mutant GgMFSD2A, in black and red, respectively. Water oxygens were considered in the intracellular cavity if within 5Å of the following residues: 51, 55, 59, 63, 64, 68, 77, 81, 85, 89, 92, 181, 185, 189, 193, 197, 201, 316, 312, 309, 305, 398, 402, 428, 432, 436, 440 or 444. f, Single-cell uptake of the fluorescent substrate LPC–NBD by cells expressing wild-type and mutant HsMFSD2A–mCherry fusion constructs. Data were normalized and represented as in b. Extended Data Figure 6 provides the raw data and n values for each construct assayed in b and f.

Extended Data Table 1 Cryo-EM data
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Supplementary information

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This file contains Supplementary Notes and Supplementary Fig. 1.

Reporting Summary

Supplementary Table 1

Raw Single Cell Flow Cytometry Data.

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Video 1

Molecular dynamics trajectory showing LPC-18:1 exploring the intracellular cavity of MFSD2A The 18:1 aliphatic tail is coloured in white, whereas the phosphate and the amine groups are coloured in red and blue, respectively. TM5 and TM8 are shown in green, while the gate residues, M182 (on TM5) and W402/F399 (on TM10), are depicted in licorice. E312 and R85 within the central charged region are drawn in surface representation and are coloured in purple. When the LPC-18:1 comes within 4Å of E312 and R85, the colour of the atoms of these residues that become engaged with the lysolipid change from purple to orange. For this video, all 24 MD trajectories with LPC-18:1 were combined into a single trajectory and the resulting trajectory was smoothened using the “smooth” Pymol (Schrödinger) function applied to a window size of 2 (corresponding to a stride of 4ns).

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Cater, R.J., Chua, G.L., Erramilli, S.K. et al. Structural basis of omega-3 fatty acid transport across the blood–brain barrier. Nature 595, 315–319 (2021). https://doi.org/10.1038/s41586-021-03650-9

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