Transport and inhibition of the sphingosine-1-phosphate exporter SPNS2

Abstract

Sphingosine-1-phosphate (S1P) is a signaling lysolipid critical to heart development, immunity, and hearing. Accordingly, mutations in the S1P transporter SPNS2 are associated with reduced white cell count and hearing defects. SPNS2 also exports the S1P-mimicking FTY720-P (Fingolimod) and thereby is central to the pharmacokinetics of this drug when treating multiple sclerosis. Here, we use a combination of cryo-electron microscopy, immunofluorescence, in vitro binding and in vivo S1P export assays, and molecular dynamics simulations to probe SPNS2's substrate binding and transport. These results reveal the transporter's binding mode to its native substrate S1P, the therapeutic FTY720-P, and the reported SPNS2-targeting inhibitor 33p. Further capturing an inward-facing apo state, our structures illuminate the protein's mechanism for exchange between inward-facing and outward-facing conformations. Finally, using these structural, localization, and S1P transport results, we identify how pathogenic mutations ablate the protein's export activity and thereby lead to hearing loss.

Fig. 1. Structure of detergent-solubilized SPNS2.

a Measurement of LMNG-purified SPNS2 by FTY720-P using nano-Differential Scanning Fluorescence (N = 3). Data are presented as mean ± SEM. b Binding affinity and kinetics of NbF09 as determined by Surface Plasmon Resonance. Injections at various concentrations and best fits are shown as orange and black lines, respectively. c Anti-FLAG immunofluorescence of SPNS2 overexpressing cells stained with FLAG-tagged NbF09 and Hoechst dye. Representative image with clear signal of the relevant immunofluorescence channel and appropriate Z plane after collecting 3–5 images from different fields of biological duplicates. d Cryo-EM map of the SPNS2-NbD12 complex determined in DDM. Transporter and nanobody are colored in purple and gray, respectively. The detergent micelle and extent of the entire molecule are shown by a thin black outline (blurred version of the same cryo-EM map). e Structure of the SPNS2-NbD12 complex determined in DDM viewed from the membrane plane. The region of the extracellular gate is indicated with a dotted box and shown in greater detail in the adjacent panel. f Residues sealing the extracellular gate of SPNS2. Hydrogen bonds are shown as dotted lines.

Fig. 2. DDM binding pocket of SPNS2.

a Bound n-dodecyl-β-D-maltopyranoside within the SPNS2 structure determined in DDM. Positive difference density of the weighted F_o − F_c difference map at 16σ and the DDM model are shown as blue surface and DDM cyan sticks, respectively. b Cross section of the SPNS2 structure, viewed from the plane of the membrane. c SPNS2 coordinates DDM through van der Waals contacts in the pocket and hydrogen bonds in the central cavity. SPNS2-DDM hydrogen bonds are shown as dotted lines. d SPNS2 N-domain contains a patch of polar residues adjacent to the DDM head group. e Schematic for GPCR-based transport assay. Media from cells expressing SPNS2 and SphK1 is collected and applied to reporter cells expressing S1PR3 and the Ca²⁺ biosensor obelin. f Measurement of S1P export activity by SPNS2 mutants (N = 24 independently treated samples). Data are presented as mean ± SEM.

Fig. 3. Substrate and inhibitor binding by SPNS2.

a Sphingosine-1-phosphate head group interactions with SPNS2 in simulation cluster 1. Carbons of SPNS2 side chains and FTY720-P are shown in purple and cyan, respectively. Interaction types are annotated by PLIP. b FTY720-P interactions with SPNS2 in simulation cluster 1. c Inhibition of S1P export by 33p measured by S1PR3-coupled export assay after incubation with 1 μM sphingosine (N = 8 independently treated samples). Data are presented as mean ± SEM. d 33p interactions within SPNS2 in simulation cluster 1. e Pose of cluster 1 for S1P, FTY720-P, and 33p with SPNS2 as viewed from the membrane plane. f Pose of cluster 1 for S1P, FTY720-P, and 33p with SPNS2 as viewed from the cytoplasm.

Fig. 4. Inward-facing apo structure of SPNS2.

a Structure of the SPNS2-NbD12 complex determined in LMNG, viewed from the plane of the membrane. The substrate and DDM binding site is indicated with a dotted box. Difference weighted F_o-F_c density maps near the DDM binding site for the SPNS2-NbD12 complex cryo-EM maps determined in (b) LMNG and (c) DDM. Maps are contoured at equivalent levels using difference density from omitted sidechains to scale the difference maps. SPNS2 in (d) inward-facing and (e) outward-facing apo states (PDB: 8EX5).

Fig. 5. Pathogenic mutations at essential locations in the SPNS2 structures.

a Locations of pathogenic SPNS2 mutations within the structure. Loop 7-8 is not resolved in the structure, and the approximate location of Pro356 is indicated by a magenta sphere with a dotted edge. b The pathogenic mutation ΔS319 within the C-domain. c Asp163 in SPNS2 is within the conserved MFS motif A and forms hydrogen bonds specific to the outward-facing conformation (PDB: 8EX5). Hydrogen bonds are shown as dotted lines. d S1P export activity by pathogenic SPNS2 mutants (N = 24 independently treated samples). Data are presented as mean ± SEM. Anti-HA immunofluorescence for HEK293-JI cells transiently transfected with HA-tagged SPNS2 (e) wild-type or mutants (f) ΔS319 and (g) D163N. Representative image with clear signal of the relevant immunofluorescence channel and appropriate Z plane after collecting 3-5 images from different fields of biological duplicates.