Figure 1.

A predicted model of SPNS2 and the binding preference of PI derivatives to full-length and truncated SPNS2

(A) The AlphaFold2 predicted model of SPNS2 (blue) with the N terminus of the full-length protein (green) shown schematically, embedded within a phospholipid bilayer. PI derivatives: PI, PI(4)P, PI(4,5)P₂, and PIP₃ are shown as purple, green, orange, and gray spheres, respectively, with binding preference to full-length and truncated SPNS2 arranged via proximity to the transporter (according to the K_D values in Table S1). The structure of S1P is also shown.

(B) Mass spectra recorded for delipidated full-length SPNS2 (5 μM) with increasing concentrations from 0 to 20 μM of PI (18:1/18:1).

(C) Plot of the mole fraction of full-length SPNS2 and PI binding calculated from titration of PI (18:1/18:1) with a resulting fit (R2 = 0.97) from a sequential ligand-binding model (solid lines). Data are plotted as mean ± standard deviation (SD) (n = 3).

(D) Plot of mole fraction of truncated SPNS2 with PI-bound states calculated from titration of PI(18:1/18:1) with a resulting fit (R2 = 0.99) from a sequential ligand-binding model (solid lines). Data are plotted as mean ± SD (n = 3).

(E) A single charge state (17+) of full-length SPNS2 following incubation with an equimolar solution containing PI, PI(4)P, PI(4,5)P₂, and PIP₃ confirms preferred binding of PI(4,5)P₂. Data are plotted as mean ± SD (n = 3). ^∗∗p < 0.01. ^∗∗∗p < 0.001. ^∗∗∗∗p < 0.0001.

(F) A single charge state (14+) of truncated SPNS2 following incubation with an equimolar solution containing PI, PI(4)P, PI(4,5)P₂, and PIP₃. Data are plotted as mean ± SD (n = 3). ^∗p < 0.05. ^∗∗p < 0.01. ^∗∗∗p < 0.001.