Fig. 4: The C-terminal Glu622/Phe623 motif blocks the intracellular cavity.
From: Synergistic activation of the human phosphate exporter XPR1 by KIDINS220 and inositol pyrophosphate
a The Glu622/Phe623 motif and the cavity-forming helices from the TMD, i.e., TMs 5, 6, 7, 8, and 10. The Glu622/Phe623 motif is colored in purple. Putative interactive residues are displayed as sticks. b 32Pi efflux of EV, WT-XPR1 and XPR1 mutants in the C-terminal plug-in loop. The numbers of biologically independent experiments are identical to (c). c 32P efflux percentages of EV and XPR1 mutants in (b) at 2 h normalized against WT-XPR1. From left to right: n = 4, 24, 7, 3, and 5 biologically independent experiments. P values are 4 × 10−4, 1, <1 × 10−4, 0.0191, and <1 × 10−4. Data are shown as mean ± s.d. in (b, c). P values were obtained by a two-tailed unpaired t-test with Welch’s correction in (c). d Western blot analysis of the binding capacity between endogenous KIDINS220 and over-expressed WT-XPR1 or XPR1 (1–630)-E622A/F623A in HEK293T cells using co-immunoprecipitation. n = 1 experiment representative of n = 2 independent transfections. Cryo-EM maps (e) and structural models (f) of the inward-facing XPR1-E622A/F623A mutant (XPR1IN) from side view and intracellular view. The color schemes are the same as in Figs. 2a, b. g The densities of Pi and the surrounding residues at the intracellular cavity are shown as blue meshes. h Structural comparison of XPR1OUT (green) and XPR1IN (light pink and light green), superimposed based on the TMD. The directions of the α3 helix backbones are indicated by arrows. The amino acid residues at the N- and C- ends of the α3 helices are labeled. Taking the α3 helix as an example, the SPX domain rotates nearly 180°.
