C2 Domain of Protein Kinase Cα:  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 Cα (PKCα) C2 domain to membranes. In the parallel model, the membrane-docking surface includes the Ca 2+ binding loops and an anion binding site on β-strands 3-4, such that the β-strands are oriented parallel to the membrane. In the perpendicular model, the docking surface is localized to the Ca 2+ binding loops and the β-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 PKCα 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, Φ, 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 β-strands 3-4 lies near the headgroups. The data support a model in which the β-strands are tilted toward the parallel orientation relative to the membrane surface. † Support provided by NIH Grant GM R01-63235 (to J.J.F.), and by DGESIC Grant PB98-0389 (to J.C.G.F.). NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author ManuscriptThe C2 domain is a conserved membrane docking motif found in numerous eukaryotic signaling proteins. The cellular functions regulated by this ubiquitous signaling domain include: (a) phosphorylation of membrane proteins, (b) production and degradation of lipid derived second messengers, (c) targeting and fusion of vesicles and membranes, (d) G protein signaling by the Ras superfamily, and (e) membrane protein ubiquitination (1-3). While sharing few sequence identities, the structural similarities between different C2 ...

Specific Translocation of Protein Kinase Cα to the Plasma Membrane Requires Both Ca2+and PIP2Recognition by Its C2 Domain

“…Biophysical studies have shown that the ␤-hairpin formed by ␤ strands 3 and 4 of the PKC␣ C2 domain lies close to the plasma membrane because of a nearly parallel orientation of the domain relative to the membrane (Kohout et al, 2003), similar to earlier predictions (Verdaguer et al, 1999). Further structural studies of PKC␣C2 complexed with Ca 2ϩ and soluble PS have revealed a basic region comprised of four lysine residues in the ␤3-4 hairpin that endow the region with a strong positive charge (Ochoa et al, 2002).…”

“…For example, our earlier work has shown that the CBLs of a related C2 domain from cPLA 2 were sufficient to promote the native intracellular targeting of this C2 domain, which docks to Golgi and ER membranes in vivo (Evans et al, 2004). Furthermore, other studies have suggested that residues located in the PKC␣ C2 domain CBLs are important in targeting of the isolated C2 domain to the plasma membrane (Conesa-Zamora et al, 2001;Stahelin et al, 2003;MarinVicente et al, 2005), and biophysical studies of PKC␣C2 docking have demonstrated that the CBLs directly contact the membrane surface (Kohout et al, 2003; Figure 1A).…”

“…Domain-containing the PKC␣ C2 Ca 2؉ -binding Loops and the ␤3-4 Hairpin A basic region formed by lysine residues in the ␤-hairpin formed by ␤ strands 3 and 4 has been postulated to coordinate PS or PIP 2 in the membrane (Ochoa et al, 2002;Corbalan-Garcia et al, 2003;Rodriguez-Alfaro et al, 2004;MarinVicente et al, 2005), and an electron paramagnetic resonance study has demonstrated that this ␤3-4 hairpin lies in close proximity to the membrane surface (Kohout et al, 2003). To investigate the contribution of the ␤3-4 hairpin to specific Figure 5.…”

“…Ca 2ϩ binding in the pocket defined by the three CBLs of the PKC␣ C2 domain has long been recognized as critical for the translocation of PKC␣ and the isolated PKC␣ C2 domain to the plasma membrane in cells and for lipid bilayer binding in vitro (Medkova and Cho, 1998;Corbalan-Garcia et al, 1999;Verdaguer et al, 1999;Conesa-Zamora et al, 2001;Stahelin and Cho, 2001;Kohout et al, 2002Kohout et al, , 2003Murray and Honig, 2002). In vitro, and presumably in vivo as well, this translocation involves binding of the Ca 2ϩ -loaded protein to anionic PS headgroups on the membrane surface (Verdaguer et al, 1999;(Evans et al, 2004).…”

Multivalent Mechanism of Membrane Insertion by the FYVE Domain

“…Although it is becoming evident that insertion of proteins into membranes is widespread, the three-dimensional orientations and quantitative binding properties remain challenging to characterize. The most common electron paramagnetic resonance and fluorescence approaches have provided important insights (17)(18)(19)(20) but require covalent attachment of paramagnetic groups to various positions of the protein or mutations of residues. The inevitable effects of these modifications on lipid interactions indicate a need to develop new methods that provide quantitative measures of bilayer insertion by native proteins.…”