Membrane docking geometry and target lipid stoichiometry of membrane-bound PKCα C2 domain: A combined molecular dynamics and experimental study

Protein kinase Cα (PKCα) possesses a conserved C2 domain (PKCα C2 domain) that acts as a Ca²⁺-regulated membrane targeting element. Upon activation by Ca²⁺, the PKCα C2 domain directs the kinase protein to the plasma membrane, thereby stimulating an array of cellular pathways. At sufficiently high Ca²⁺ concentrations, binding of the C2 domain to the target lipid phosphatidylserine (PS) is sufficient to drive membrane association; however, at typical physiological Ca²⁺ concentrations, binding to both PS and phosphoinositidyl-4,5-bisphosphate (PIP₂) is required for specific plasma membrane targeting. Recent EPR studies have revealed the membrane docking geometries of the PKCα C2 domain docked to (i) PS alone and (ii) both PS and PIP₂ simultaneously. These two EPR docking geometries exhibit significantly different tilt angles relative to the plane of the membrane, presumably induced by the large size of the PIP₂ headgroup. The present study utilizes the two EPR docking geometries as starting points for molecular dynamics simulations that investigate atomic features of the protein-membrane interaction. The simulations yield approximately the same PIP₂-triggered change in tilt angle observed by EPR. Moreover, the simulations predict a PIP₂:C2 stoichiometry approaching 2:1 at a high PIP₂ mole density. Direct binding measurements titrating the C2 domain with PIP₂ in lipid bilayers yield a 1:1 stoichiometry at moderate mole densities and a saturating 2:1 stoichiometry at high PIP₂ mole densities. Thus, the experiment confirms the target lipid stoichiometry predicted by EPR-guided molecular dynamics simulations. Potential biological implications of the observed docking geometries and PIP₂ stoichiometries are discussed.