The Pleckstrin Homology Domain of Phospholipase C-δ₁ Binds with High Affinity to Phosphatidylinositol 4,5-Bisphosphate in Bilayer Membranes

The pleckstrin homology (PH) domain of phospholipase C-δ₁ (PLC-δ₁) binds to phosphatidylinositol 4,5-bisphosphate (PI(4,5)P₂) in phospholipid membranes with an affinity (K_a ~ 10⁶ M^-1) and specificity comparable to those of the native enzyme. PLC-δ₁ and its PH domain also bind inositol 1,4,5-trisphosphate, the polar head group of PI(4,5)P2, with comparable affinity and approximately 1:1 stoichiometry. A peptide corresponding to amino acids 30-43 of the PLC-δ₁ PH domain contains several basic residues predicted to bind PI(4,5)P₂, but binds weakly and with little specificity for PI(4,5)P₂; hence the tertiary structure of the isolated PH domain is required for high affinity PI(4,5)P₂ binding. Our PI-(4,5)P₂ binding results support the hypothesis that the intact PH domain, serving as a specific tether, directs PLC-δ₁ to membranes enriched in PI(4,5)P₂ and permits the active site, located elsewhere in the protein, to hydrolyze multiple substrate molecules before this enzyme dissociates from the membrane surface.