Fig. 4: TMD of AE2 in complex with PIP₂.

a Structural comparison of the PIP₂-bound TMDs of inward-facing AE2_IF/PIP2 and outward-facing AE1_OF/PIP2 (8CT3) aligned by the gate domain. The gate domain and core domain of AE2 are colored in blue and green, respectively. AE1_OF/PIP2 is colored in gray. b, c Structural comparison of one protomer of the AE1_OF/PIP2 TMD (gray) and the AE2_IF/PIP2 TMD (colored) viewed from the side (b) and from the extracellular side (c). Displacements of the core domain from outward-facing AE1_OF/PIP2 to inward-facing AE2_IF/PIP2 are indicated by red arrows. d, e Comparison of the PIP₂ binding site of AE2_IF/PIP2 (d) and AE1_OF/PIP2 (e). PIP₂, residues formed the PIP₂ binding site of AE2_IF/PIP2 and the corresponding residues in AE1_OF/PIP2 are shown as sticks. f Thermal shift stabilization of mCherry-tagged wild-type AE2 (blue) and AE2 with mutations in the PIP₂ binding sites (red). The mCherry fluorescence of AE2 dimer peak after heating was normalized to that without heating. The data are presented as mean values ± SD. The number of independent experiments for both wild-type AE2 and triple mutant AE2-R932A/K1174A/H1148A is 3. The Tm₅₀ values for wild-type AE2 and triple mutant AE2 are 54.0 °C and 53.5 °C, respectively. The apparent melting temperature Tm was calculated using a sigmoidal four-parameter logistic regression function and a p value of 0.24 (not significant) was determined by a two-sided F-test. g Thermal shift stabilization of wild-type AE1 (blue) and AE1 with disrupted PIP₂ binding sites (red). The data are normalized fluorescence and presented as mean values ± SD as in f (n = 3 independent experiments for both wild-type and triple mutant AE1). The Tm₅₀ values for wild-type AE1 and AE1-R602A/K817A/Y818A are 57.2 °C and 60.4 °C, respectively. The apparent melting temperature Tm was calculated using a sigmoidal four-parameter logistic regression function and a p value of 2.15× 10⁻⁷ (<0.05; significant) was determined by a two-sided F-test.