title: C2 domain of protein kinase C alpha: elucidation of the membrane docking surface by site-directed fluorescence and spin labeling.
abstract: The C2 domain is a conserved signaling motif that triggers membrane docking in a Ca(2+)-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 C alpha (PKC alpha) C2 domain to membranes. In the parallel model, the membrane-docking surface includes the Ca(2+) 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 Ca(2+) 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 PKC alpha 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 Ca(2+) activation or membrane docking trigger measurable fluorescence changes. Ca(2+) binding was found to initiate a global conformational change, while membrane docking triggered the largest fluorescein environmental changes at labeling positions on the three Ca(2+) 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 Ca(2+) 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 Ca(2+) first and third Ca(2+) 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.
authors.affiliation: Department of Chemistry and Biochemistry, University of Colorado, Boulder, Colorado 80309-0215, USA.
authors.forename: Susy C
authors.initials: SC
authors.lastname: Kohout
authors.forename: Senena
authors.initials: S
authors.lastname: Corbalán-García
authors.forename: Juan C
authors.initials: JC
authors.lastname: Gómez-Fernández
authors.forename: Joseph J
authors.initials: JJ
authors.lastname: Falke
mesh_terms: D019943:Amino Acid Substitution / Q000235:genetics; D002118:Calcium / Q000737:chemistry; D003497:Cyclic N-Oxides / Q000737:chemistry; D003545:Cysteine / Q000235:genetics; D004492:Edetic Acid / Q000031:analogs & derivatives* / Q000737:chemistry; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods; D019793:Fluorescein / Q000737:chemistry; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods; D008563:Membrane Lipids / Q000737:chemistry; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding / Q000235:genetics; D011487:Protein Conformation; D011493:Protein Kinase C / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D051571:Protein Kinase C-alpha; D017434:Protein Structure, Tertiary / Q000235:genetics; D013113:Spin Labels; D019097:Spin Trapping / Q000379:methods; D013499:Surface Properties
chemical_list: D003497:Cyclic N-Oxides; D008563:Membrane Lipids; D008565:Membrane Proteins; D013113:Spin Labels; C012517:proxyl nitroxide; C026058:EDDA; D004492:Edetic Acid; D011493:Protein Kinase C; D051571:Protein Kinase C-alpha; D003545:Cysteine; D002118:Calcium; D019793:Fluorescein