C2 domain of protein kinase Cα:: Elucidation of the membrane docking surface by site-directed fluorescence and spin labeling

The C2 domain is a conserved signaling motif that triggers membrane docking in a Ca2+- dependent manner, but the membrane docking surfaces of many C2 domains have not yet been identified. Two extreme models can be proposed for the docking of the protein kinase Calpha (PKCalpha) C2 domain to membranes. In the parallel model, the membrane-docking surface includes the Call binding loops and an anion binding site on beta-strands 3-4, such that the beta-strands are oriented parallel to the membrane. In the perpendicular model, the docking surface is localized to the Call binding loops and the beta-strands are oriented perpendicular to the membrane surface. The present study utilizes site-directed fluorescence and spin-labeling to map out the membrane docking surface of the PKCalpha C2 domain. Single cysteine residues were engineered into 18 locations scattered over all regions of the protein surface, and were used as attachment sites for spectroscopic probes. The environmentally sensitive fluorescein probe identified positions where Ca2+ activation or membrane docking trigger measurable fluorescence changes. Ca2+ binding was found to initiate a global conformational change, while membrane docking triggered the largest fluorescein environmental changes at labeling positions on the three Ca2+ binding loops (CBL), thereby localizing these loops to the membrane docking surface. Complementary EPR power saturation measurements were carried out using a nitroxide spin probe to determine a membrane depth parameter, Phi, for each spin-labeled mutant. Positive membrane depth parameters indicative of membrane insertion were found for three positions, all located on the Ca2+ binding loops: N189 on CBL 1, and both R249 and R252 on CBL 3. In addition, EPR power saturation revealed that five positions near the anion binding site are partially protected from collisions with an aqueous paramagnetic probe, indicating that the anion binding site lies at or near the surface of the headgroup layer. Together, the fluorescence and EPR results indicate that the Ca2+ first and third Ca2+ binding loops insert directly into the lipid headgroup region of the membrane, and that the anion binding site on beta-strands 3-4 lies near the headgroups. The data support a model in which the beta-strands are tilted toward the parallel orientation relative to the membrane surface.