Introduction
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
Discussion
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
REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Mouse monoclonal anti-FLAG tag antibody MBL International Cat# M185-3L; RRID:AB_11123930 Mouse monoclonal anti-mCherry antibody Novus Biologicals Cat# NBP1-96752SS; RRID:AB_11008969 Mouse monoclonal anti-Actin antibody Santa Cruz Biotechnology Cat# sc-8432; RRID: AB_626630 Anti-mouse IgG, HRP-linked antibody Cell Signaling Technology Cat# 7076; RRID:AB_330924 Bacterial and virus strains E.coli DH5α Competent Cells GoldBio Cat# CC-101-TR E.coli DH10Bac Competent
Acknowledgments
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
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