Fig. 6.

WHAMM binds to PI(4,5)P₂ through two conserved amphipathic helixes. a Flotation assays to identify the membrane-binding regions in WHAMM beyond the originally identified WMD (1-260) domain. Liposomes with only DOPC-DOPE were used as the control. b Comparison of the domain structure of WHAMM based on a previous model (top) and proposed in current work (bottom). Two regions suspected to be responsible for the membrane interaction of WHAMM are highlighted in purple. c Sequence alignment of helix 1 and helix 2, along with their secondary structure prediction³⁹. The indicated point mutations (arrows) were introduced at the sites highlighted in red. The dashed box shows the region used to generate the helical wheel in the next panel. d Corresponding helical wheels of helix 1 and helix 2 generated using HELIQUEST⁴⁰. The hydrophobic face is highlighted as a light blue sector. The black arrows indicate the direction of hydrophobic moments. The mutations that disrupt the hydrophobic face are also shown. e FL WHAMM and fragments containing helix 1 (1-260) or helix 2 (310-809) with and without the indicated mutations were purified and incubated with liposomes containing DOPC-DOPE only or with 15% PI(4,5)P₂ and applied to the flotation assay. Each fraction was collected carefully, then proteins were separated by SDS-PAGE and visualized using Coomassie blue. f Band density in the top fraction from each experiment in e was measured and normalized with total input using ImageJ. The proportion of protein bound to liposomes was compared between the mutants and WT from two or three independent experiments. Error bars indicate SEM. For 1-260 (n = 3), results were compared using one-way ANOVA followed with Holm-Sidak’s multiple comparisons test. ***p < 0.001. For 310-809 (n = 2) and FL (n = 2). Source data are provided as a Source Data file