Membrane docking of the C2 domain from protein kinase Cα as seen by polarized ATR-IR. The role of PIP2

We have used attenuated total internal reflection infrared spectroscopy (ATR-IR) spectroscopy to study the association of the C2 domain from protein kinase C alpha (PKC alpha) with different phospholipid membranes, so as to characterise the mode of membrane docking and its modulation by the second-messenger lipid PIP2. In parallel, we have also examined the membrane interaction of the C2 domain from cytosolic phospholipase A(2). PIP2 did not induce significant changes in secondary structure of the membrane-bound PKC alpha-C2 domain, nor did binding of the PKC alpha-C2 domain change the dichroic ratios of the lipid chains, whereas the C2 domain from phospholipase A(2) did perturb the lipid chain orientation. Measurements of the dichroic ratios for the amide I and amide II protein bands were combined so as to distinguish the tilt of the beta-sheets from that of the beta-strands within the sheet. When associated with POPC/POPS membranes, the beta-sandwich of the PKC alpha-C2 domain is inclined at an angle alpha = 35 degrees to the membrane normal, i.e., is oriented more nearly perpendicular than parallel to the membrane. In the process of membrane docking, the tilt angle increases to alpha = 44 degrees in the presence of PIP2, indicating that the beta-sandwich comes closer to the membrane surface, so confirming the importance of this lipid in determining docking of the C2 domain and consequent activation of PKC alpha. (c) 2010 Elsevier B.V. All rights reserved.