Structural and functional insights into Spns2-mediated transport of sphingosine-1-phosphate (2023)


Sphingosine-1-phosphate (S1P) regulates the immune system, angiogenesis, auditory function, and epithelial and endothelial barrier integrity.1,2,3,4,5 Two sphingosine kinase (SphK) isoenzymes, SphK1 and SphK2, catalyze the phosphorylation of sphingosine (Sph) to produce S1P in cells.6 In turn, the intracellularly synthesized S1P must be exported from cells to enter circulatory fluids and activate its receptors for downstream signaling. Two major facilitator superfamily (MFS) transporters are involved in S1P export—spinster homolog 2 (Spns2) primarily exports S1P in endothelial cells,7,8,9 whereas Mfsd2b functions in erythrocytes and platelets.10,11 Spns2 was the first identified mammalian S1P transporter,12,13,14 and it plays an essential role in the lymphatic system, supplying lymph S1P and enabling lymphocyte circulation. Notably, Spns2 also transports non-natural S1P analogs, including FTY720 phosphate,15 a clinical drug used to treat multiple sclerosis.16,17

The physiological importance of Spns2 is further supported by studies in Spns2-knockout mice, which show rapid loss of auditory sensitivity and complete deafness before 3weeks of age18 and aberrant lymphatic sinuses in the lymph nodes.9 The absence of Spns2 impairs the postnatal retinal morphogenesis.19 Interestingly, Spns2 deficiency protects mice from the development of multiple sclerosis and other autoimmune diseases20,21 and reduces pulmonary metastasis.22 These findings uncover pivotal functions of Spns2 in cancer, the auditory system, ocular development, and inflammatory and autoimmune diseases.6,23 Therefore, pharmacological modulation of Spns2 has considerable therapeutic potential.

The molecular mechanism of how Spns2 transports S1P remains poorly understood. To date, five MFS lysolipid transporters—Spns1, Spns2, Mfsd2a, Mfsd2b, and bacterial LplT—have been shown to transport amphiphilic lysolipids,24,25 but there is a paucity of structural information. Recently, structures of Mfsd2a, which transports omega-3 fatty acid across the blood-brain barrier, have been reported in its inward- and outward-facing states.26,27,28 Although one can expect Spns2 to have an overall architecture similar to that of Mfsd2a, insights gained from structures of the latter cannot be readily extended to Spns2, as these two transporters also share low sequence identity and have distinct substrate preferences. They also differ in transport mechanisms: Mfsd2a imports lysophosphatidylcholine (LPC) into cells in a Na+-dependent manner,29 whereas Spns2 was proposed to be a proton-coupled30 or a facilitated-diffusion S1P exporter.31 Thus, structural information on Spns2 is required to understand this important transporter.

Here, we capture structures of human Spns2 in multiple functionally relevant states, at resolutions of up to 2.9Å, using single-particle cryo-electron microscopy (cryo-EM). The structures illuminate the S1P export cycle, revealing two intermediate conformations that connect the inward- and outward-facing states, which had not been previously captured for other MFS lipid transporters. Furthermore, we determine a structure that uncovers the inhibitory mechanism of the Spns2-specific inhibitor, 16d, described very recently.32 Our structural and functional analyses elucidate the transport process of S1P via Spns2 and provide a better understanding of S1P metabolism and signaling.

Section snippets

Structure determination of Spns2

Spns2 is a small membrane protein (∼58kDa), with most of its mass embedded in the membrane. The small size and lack of clearly distinguishable features protruding out of the membrane make cryo-EM particle alignment challenging for Spns2. To overcome these obstacles, we fused a maltose-binding protein (MBP) to the N terminus of Spns2 to increase the particle size and to allow accurate particle alignment. Specifically, we connected the C-terminal helix of MBP to the N terminus of the first


In this study, we report four different functional states of the human S1P transporter Spns2. The S1P substrate is accommodated by an inward-facing cavity, with its alkyl tail interacting with hydrophobic residues in TMs 7, 8, and 10 (Figure3B). Structural analysis reveals an asymmetric rearrangement in Spns2 during alternating-access transport (Figure4B). Hydrophilic interactions between charged residues in the N- and C-domains contribute to the rocker-switch-type movement of both domains,

Key resources table

Mouse monoclonal anti-FLAG tag antibodyMBL InternationalCat# M185-3L; RRID:AB_11123930
Mouse monoclonal anti-mCherry antibodyNovus BiologicalsCat# NBP1-96752SS; RRID:AB_11008969
Mouse monoclonal anti-Actin antibodySanta Cruz BiotechnologyCat# sc-8432; RRID: AB_626630
Anti-mouse IgG, HRP-linked antibodyCell Signaling TechnologyCat# 7076; RRID:AB_330924
Bacterial and virus strains
E.coli DH5α Competent CellsGoldBioCat# CC-101-TR
E.coli DH10Bac Competent


The cryo-EM data were collected at the Cryo-EM Center of the St. Jude Children’s Research Hospital and at the UT Southwestern Medical Center Cryo-EM Facility (funded in part by the CPRIT Core Facility Support Award RP170644). We thank Y. Wang, L. Esparza, and L. Beatty for cell culture. We thank I. Chen and E. Debler for editing the manuscript, and we thank J. Saunders and J. Fortanet for 16d synthesis. This work was supported by NIH P01 HL160487 and 1P30DK127984 (to J.G.M.), NIH R01 GM135343

Recommended articles (6)

  • Research article

    Plasma membrane preassociation drives β-arrestin coupling to receptors and activation

    Cell, Volume 186, Issue 10, 2023, pp. 2238-2255.e20

    β-arrestin plays a key role in G protein-coupled receptor (GPCR) signaling and desensitization. Despite recent structural advances, the mechanisms that govern receptor-β-arrestin interactions at the plasma membrane of living cells remain elusive. Here, we combine single-molecule microscopy with molecular dynamics simulations to dissect the complex sequence of events involved in β-arrestin interactions with both receptors and the lipid bilayer. Unexpectedly, our results reveal that β-arrestin spontaneously inserts into the lipid bilayer and transiently interacts with receptors via lateral diffusion on the plasma membrane. Moreover, they indicate that, following receptor interaction, the plasma membrane stabilizes β-arrestin in a longer-lived, membrane-bound state, allowing it to diffuse to clathrin-coated pits separately from the activating receptor. These results expand our current understanding of β-arrestin function at the plasma membrane, revealing a critical role for β-arrestin preassociation with the lipid bilayer in facilitating its interactions with receptors and subsequent activation.

  • Research article

    Rational exploration of fold atlas for human solute carrier proteins

    Structure, Volume 30, Issue 9, 2022, pp. 1321-1330.e5

    The solute carrier (SLC) superfamily is the largest group of proteins responsible for the transmembrane transport of substances in human cells. It includes more than 400 members that are organized into 65 families according to their physiological function and sequence similarity. Different families of SLCs can adopt the same or different folds that determine the mechanism and reflect the evolutionary relationship between SLC members. Analysis of structural data in the literature before this work showed 13 different folds in the SLC superfamily covering 40 families and 343 members. To further study their mechanism, we systematically explored the SLC superfamily to look for more folds. Based on our results, at least three new folds are found for the SLC superfamily, one of which is in the choline-like transporter family (SLC44) and has been experimentally verified. Our work has laid a foundation and provided important insights for the systematic and comprehensive study of the structure and function of SLC.

  • Research article

    Structures of LRP2 reveal a molecular machine for endocytosis

    Cell, Volume 186, Issue 4, 2023, pp. 821-836.e13

    The low-density lipoprotein (LDL) receptor-related protein 2 (LRP2 or megalin) is representative of the phylogenetically conserved subfamily of giant LDL receptor-related proteins, which function in endocytosis and are implicated in diseases of the kidney and brain. Here, we report high-resolution cryoelectron microscopy structures of LRP2 isolated from mouse kidney, at extracellular and endosomal pH. The structures reveal LRP2 to be a molecular machine that adopts a conformation for ligand binding at the cell surface and for ligand shedding in the endosome. LRP2 forms a homodimer, the conformational transformation of which is governed by pH-sensitive sites at both homodimer and intra-protomer interfaces. A subset of LRP2 deleterious missense variants in humans appears to impair homodimer assembly. These observations lay the foundation for further understanding the function and mechanism of LDL receptors and implicate homodimerization as a conserved feature of the LRP receptor subfamily.

  • Research article

    PERIOD phosphorylation leads to feedback inhibition of CK1 activity to control circadian period

    Molecular Cell, Volume 83, Issue 10, 2023, pp. 1677-1692.e8

    PERIOD (PER) and Casein Kinase 1δ regulate circadian rhythms through a phosphoswitch that controls PER stability and repressive activity in the molecular clock. CK1δ phosphorylation of the familial advanced sleep phase (FASP) serine cluster embedded within the Casein Kinase 1 binding domain (CK1BD) of mammalian PER1/2 inhibits its activity on phosphodegrons to stabilize PER and extend circadian period. Here, we show that the phosphorylated FASP region (pFASP) of PER2 directly interacts with and inhibits CK1δ. Co-crystal structures in conjunction with molecular dynamics simulations reveal how pFASP phosphoserines dock into conserved anion binding sites near the active site of CK1δ. Limiting phosphorylation of the FASP serine cluster reduces product inhibition, decreasing PER2 stability and shortening circadian period in human cells. We found that Drosophila PER also regulates CK1δ via feedback inhibition through the phosphorylated PER-Short domain, revealing a conserved mechanism by which PER phosphorylation near the CK1BD regulates CK1 kinase activity.

  • Research article

    Negative allosteric modulation of the glucagon receptor by RAMP2

    Cell, Volume 186, Issue 7, 2023, pp. 1465-1477.e18

    Receptor activity-modifying proteins (RAMPs) modulate the activity of many Family B GPCRs. We show that RAMP2 directly interacts with the glucagon receptor (GCGR), a Family B GPCR responsible for blood sugar homeostasis, and broadly inhibits receptor-induced downstream signaling. HDX-MS experiments demonstrate that RAMP2 enhances local flexibility in select locations in and near the receptor extracellular domain (ECD) and in the 6th transmembrane helix, whereas smFRET experiments show that this ECD disorder results in the inhibition of active and intermediate states of the intracellular surface. We determined the cryo-EM structure of the GCGR-Gs complex at 2.9Å resolution in the presence of RAMP2. RAMP2 apparently does not interact with GCGR in an ordered manner; however, the receptor ECD is indeed largely disordered along with rearrangements of several intracellular hallmarks of activation. Our studies suggest that RAMP2 acts as a negative allosteric modulator of GCGR by enhancing conformational sampling of the ECD.

  • Research article

    Structure of Semliki Forest virus in complex with its receptor VLDLR

    Cell, Volume 186, Issue 10, 2023, pp. 2208-2218.e15

    Semliki Forest virus (SFV) is an alphavirus that uses the very-low-density lipoprotein receptor (VLDLR) as areceptor during infection of its vertebrate hosts and insect vectors. Herein, we used cryoelectron microscopy to study the structure of SFV in complex with VLDLR. We found that VLDLR binds multiple E1-DIII sites of SFV through its membrane-distal LDLR class A (LA) repeats. Among the LA repeats of the VLDLR, LA3 has the best binding affinity to SFV. The high-resolution structure shows that LA3 binds SFV E1-DIII through a small surface area of 378Å2, with the main interactions at the interface involving salt bridges. Compared with the binding of single LA3s, consecutive LA repeats around LA3 promote synergistic binding to SFV, during which the LAs undergo a rotation, allowing simultaneous key interactions atmultiple E1-DIII sites on the virion and enabling the binding of VLDLRs from divergent host species to SFV.

© 2023 Elsevier Inc.


Top Articles
Latest Posts
Article information

Author: Fredrick Kertzmann

Last Updated: 09/16/2023

Views: 6031

Rating: 4.6 / 5 (46 voted)

Reviews: 93% of readers found this page helpful

Author information

Name: Fredrick Kertzmann

Birthday: 2000-04-29

Address: Apt. 203 613 Huels Gateway, Ralphtown, LA 40204

Phone: +2135150832870

Job: Regional Design Producer

Hobby: Nordic skating, Lacemaking, Mountain biking, Rowing, Gardening, Water sports, role-playing games

Introduction: My name is Fredrick Kertzmann, I am a gleaming, encouraging, inexpensive, thankful, tender, quaint, precious person who loves writing and wants to share my knowledge and understanding with you.