BPS2026 – Cholesterol and PE influence PIP dynamics in model membranes containing PC and PS

Phosphatidylinositol phosphates (PIPs) are anionic phospholipids that constitute a minor portion of eukaryotic membranes. PIPs regulate cellular processes through specific interactions with numerous membrane proteins, despite their low abundance. Previous studies have probed the interactions of isolated PIPs with membrane proteins, but the mechanisms of binding and activation remain unclear. Here, we investigate the dynamics of phosphatidylinositol (3,4,5) trisphosphate (PIP 3 ) in model membranes containing phosphatidylethanolamine (PE), phosphatidylcholine (PC), phosphatidylserine (PS) and cholesterol to closely mimic complex membrane environments. Solid-state NMR (ssNMR) and molecular dynamics (MD) simulations are employed to monitor the individual and cumulative effects of PE and cholesterol on PIP 3 in PIP 3 /PC/PS bilayers. Liposomes are made using thin film hydration, followed by sonication and extrusion. ssNMR data show increased lipid tail ordering upon addition of cholesterol and/or PE. MD simulations further reveal PIP 3 dimer formation, enhanced in cholesterol-containing membranes, with cholesterol positioned between PIP3 dimers and existing either as a monomer or a dimer. We also observe intramolecular hydrogen bonding between phosphate groups and hydroxyl groups on the inositol ring, with the ring adopting two main conformations that alter its hydrogen-bonding pattern. These results indicate that membrane composition significantly affects PIP 3 clustering, tail ordering, and conformational dynamics in membranes, which may be crucial for modulating PIP-protein interactions and downstream signaling.