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

Abstract

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.

Structural insights into the Ca 2+ and PI(4,5)P 2 binding modes of the C2 domains of rabphilin 3A and synaptotagmin 1

“…Given the close proximity of the lysine-rich cluster and the CBR, these results are compatible with a parallel orientation model (Fig. 6F) in which these two motifs would interact with a vesicle representing the plasma membrane, leaving the bottom face free to interact in trans with another lipid vesicle (19,(47)(48)(49)(50)(51). How these two C2 domains act in tandem and in cooperation with other SNARE proteins to regulate vesicle fusion is still under debate and will need further exploration.…”

The molecular basis of ceramide-1-phosphate recognition by C2 domains

“…The stoichiometric ratio of C1P to cPLA 2 ␣ -C2 was determined through modifi cation of the previously established methodology as described for the C2 domain of PKC ␣ and PI(4,5)P 2 ( 37 ). HEPES, pH 7.4, 10 mM, containing 0.16 M KCl and 500 nM Ca 2+ , where and catalytic domain, respectively.…”

“…Subsequently, small unilamellar vesicles containing POPC-POPE-C1P (50:40:10) were added at increasing molar C1P concentrations until the fl uorescence was no longer quenched. The data were normalized, where the maximum tryptophan quenched was set to 1, then the rise and saturation phases of the data were fi t using a linear least-squares equation ( 37 ). The intersection of the two lines defi nes the stoichiometry of cPLA 2 ␣ -C2 to C1P.…”

“…As vesicles containing POPC-POPE-C1P (50:40:10) were titrated into a fi xed concentration of cPLA 2 ␣ -C2, quenching was measured until no further change was detectable. The stoichiometry of binding was calculated as previously described by fi tting the rise and saturation phases of the fl uorescence data ( 37 ). The intersection of the lines gave a ratio of 1:1.16 ± 0.13, which is approximately 1 C1P to 1 cPLA 2 ␣ -C2 ( Fig.…”

Electrostatic and Hydrophobic Interactions Differentially Tune Membrane Binding Kinetics of the C2 Domain of Protein Kinase Cα

“…Thus, Ca 2ϩ serves a specific structural role in allowing the C2 domain to bind anionic membranes. In addition to the Ca 2ϩ -binding site, a lysine-rich cluster within the ␤3-and ␤4-sheets of the C2 domain binds phosphatidylinositol 4,5-bisphosphate (27,(32)(33)(34)(35)(36). It is this interaction with phosphatidylinositol 4,5-bisphosphate that directs conventional PKC isozymes to the plasma membrane, which is enriched in this lipid (38 -41).…”