Membrane specificity of the human cholesterol transfer protein STARD4

[Display omitted] •The binding and orientation of human STARD4 on lipid bilayers depends on both the presence of the cargo, and on the lipid composition.•The binding of apo human STARD4 is favored by the presence of PIP2, through two PIP2 binding sites on the protein surface.•Holo STARD4 is less sensitive to lipid composition than the apo form.•Simulations provide two new structure models of STARD4: a closed holo conformation where Ω1 is displaced, and an open gate in the presence of cholesterol where Ω4 is displaced.•Our study supports the capacity of human STARD4 to achieve directed transfer between organelle membranes. STARD4 regulates cholesterol homeostasis by transferring cholesterol between the plasma membrane and endoplasmic reticulum. The STARD4 structure features a helix-grip fold surrounding a large hydrophobic cavity holding the sterol. Its access is controlled by a gate formed by the Ω1 and Ω4 loops and the C-terminal α-helix. Little is known about the mechanisms by which STARD4 binds to membranes and extracts/releases cholesterol. All available structures of STARD4 are without a bound sterol and display the same closed conformation of the gate. The cholesterol transfer activity of the mouse STARD4 is enhanced in the presence of anionic lipids, and in particular of phosphatidylinositol biphosphates (PIP2) for which two binding sites were proposed on the mouse STARD4 surface. Yet only one of these sites is conserved in human STARD4. We here report the results of a liposome microarray-based assay and microseconds-long molecular dynamics simulations of human STARD4 with complex lipid bilayers mimicking the composition of the donor and acceptor membranes. We show that the binding of apo form of human STARD4 is sensitive to the presence of PIP2 through two specific binding sites, one of which was not identified on mouse STARD4. We report two novel conformations of the gate in holo-STARD4: a yet-unobserved close conformation and an open conformation of Ω4 shedding light on the opening/closure mechanism needed for cholesterol uptake/release. Overall, the modulation of human STARD4 membrane-binding by lipid composition, and by the presence of the cargo supports the capacity of human STARD4 to achieve directed transfer between specific organelle membranes.