Cooperativity of PIP2 and PS lipids modulates PH domain binding

Phosphatidylinositides constitute only 1%-3% of plasma membranes but play vital roles in cellular signaling. In particular, phosphatidylinositol 4,5-bisphosphate (PIP₂) is involved in processes such as cytoskeleton organization and ion-channel regulation. Pleckstrin homology (PH) domains are modular domains found in many proteins and are known for their strong affinity for PIP₂ headgroups. The role of lipid composition in PH domain binding to PIP₂, particularly the inclusion of phosphatidylserine (PS), is not well understood. This study explores the mechanisms of PH domain binding to PIP₂ using fluorescence spectroscopy, Fourier transform infrared spectroscopy, two-dimensional infrared spectroscopy, and molecular dynamics simulations. We find that anionic PIP₂ and PS alter the interfacial environment compared to phosphatidylcholines. Additionally, the PH domain promotes the localization of anionic lipid domains upon binding. Our results highlight the role of PS in lipid domain formation within membranes and its potential influence on protein binding affinities and lipid geometries. Specifically, we discovered a strong interaction between PIP₂ and PS whereby hydrogen bonding within these anionic lipids drives localization in the membrane. This interaction also regulates protein binding at the membrane interface. Our findings suggest that cooperativity between PIP₂ and PS is key to the formation of localized lipid domains and the recruitment of proteins such as the PH domain of phospholipase C-δ1.