The selective cargo loading and release functions of the cellular sterol transporter StarD4 are allosterically controlled by the recognition of specific PIP2 subtypes in the targeted membranes

Intracellular cholesterol trafficking, vital for cell function, is orchestrated in part by proteins with steroidogenic acute regulator-related lipid transfer (START) domains, such as StarD4 which is a sterol-specific transfer protein that contributes ∼ 33% of non-vesicular sterol transport between plasma membrane (PM), the primary cholesterol reservoir, and the endoplasmic reticulum (ER). Comprising a single soluble START domain, StarD4 is known to target both sterol donor-, and acceptor-membranes, through interactions with anionic lipids. Experiments have illuminated the kinetics of this sterol transfer and shown it to be modulated by specific phosphatidylinositol phosphates (PIPs) on the target membrane. The distinct subtype distribution of PIPs in the membranes of cellular organelles serves as a guide directing StarD4 to particular cell components. To discover the molecular mechanism of membrane targeting by the recognition of the PIP2 subtype in the membrane, and how this affects the direction and kinetics of CHL transport (uptake vs release), we used molecular dynamics (MD) to simulate the processes. Analysis of the MD trajectories with machine learning and information theory methods revealed how StarD4 embeds in membrane containing different anionic lipids (PI(4,5)P ₂ , PI(3,5)P ₂ , and PS), how it recognizes the anionic lipid content by the different modes in which they bind to the protein, and how the protein responds to this recognition by adopting different orientations on the membrane and undergoing specific conformational changes. We identified the allosteric channels underlying these complex dynamics rearrangements by connecting the various PIP2-subtype-specific conformational states to different CHL binding modes in the pocket, which then relate to the dynamics of the gates that allow either CHL uptake into apo -StarD4, or its release from holo- StarD4. This reveals the crucial role of PIP2 subtypes in shaping functional StarD4 motifs responsible for organelle selectivity of the cholesterol trafficking, providing fundamental insights into cellular cholesterol regulation.