Fig. 1: Cryo-EM structure of S1P bound Spns2 and biochemical characterization of Spns2-mediated S1P transport.

a Deficiency of Spns2 induced two-heart phenotypes of zebrafish. “–” represents the normal heart phenotype; “c”, “+”, “++”, and “+++” represent the severity levels of two-heart phenotypes. b Inward-open structure of Spns2_fusion. TMs of the NTD and CTD are colored pale green and salmon, respectively. The long flexible cytoplasmic loop (P282–S301) between NTD and CTD is colored gray. An elongated density in the central cavity is shown in mesh and fitted with an S1P molecule. S1P is shown as sticks and colored yellow. c, d Residues (S326, Q463, S464) coordinated the hydrophilic group of S1P in the structure of Spns2_fusion. Residues (Y246 and I429) interact with the tail of S1P. S1P is shown as b. e S1P transport assay of WT Spns2 and Y246A, I429A, and S326A-Q463A-S464A mutants. Data are presented as mean ± SD. n ≥ 6 biological replicates; ****P < 0.0001. f Statistics of two heart ratios of the WT Spns2 and Y246A, I429A, and S326A-Q463A-S464A mutants in the zebrafish two-heart rescue assay. The different two-heart phenotypes are counted and referred to as a. The severity levels of two-heart phenotypes are shown in different colors. The total embryo numbers of each group are labeled at the top of each histogram. g Four positively charged residues (R227, R456, R119, and R200) line up in the central cavity. Residues are shown as sticks. A cut view of the central cavity with electrostatic potential is displayed. h Residues include D118, R119, R200, and E207 on the NTD side. Residues are shown as sticks. R119 interacts with E207 via a salt bridge labeled by a dashed line. i Critical interactions at the extracellular side of Spns2 in the inward-open structure. A salt bridge formed between D137 and R342, and interaction between D128 and K253 stabilize the close state of the extracellular side. Residues are shown as sticks. j S1P transport assay of WT Spns2 and different mutants. Data are presented as mean ± SD. n ≥ 4 biological replicates, ****P < 0.0001. k Statistics of two heart ratios of the WT Spns2 and mutants in the zebrafish two-heart rescue assay shown as f. l Schematic of Spns2-mediated S1P transport cycle. The NTD and the CTD are colored pale green and salmon, respectively. Key residues are displayed and labeled. S1P is colored yellow. Two presented structures represent two conformations in red boxes. The reported structure (PDB: 8EX5) represents the conformation in the gray box.