Membrane Docking Geometry and Target Lipid Stoichiometry of Membrane-Bound PKCα C2 Domain: A Combined Molecular Dynamics and Experimental Study

摘要

Protein kinase Ca (PKCα) possesses a conserved C2 domain (PKCα C2) that acts as a Ca²⁺-regulated membrane targeting element. Upon activation by Ca²⁺, PKCα C2 directs the kinase protein to the plasma membrane, thereby stimulating an array of cellular pathways. At sufficiently high Ca²⁺ concentrations the binding of the C2 domain to the target lipid phosphatidylserine (PS) is sufficient to drive membrane association, but at typical physiological Ca²⁺ concentrations binding both to PS and to phosphoinositidyl-4,5-bisphosphate (PIP2) is required for specific plasma membrane targeting. Recent EPR studies have revealed the membrane docking geometries of PKCα C2 docked to (i) PS alone, and to (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 the 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 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, 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.