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

Biochim Biophys Acta. 2011 Mar;1808(3):684-95. doi: 10.1016/j.bbamem.2010.11.035. Epub 2010 Dec 7.

Abstract

We have used attenuated total internal reflection infrared spectroscopy (ATR-IR) spectroscopy to study the association of the C2 domain from protein kinase Cα (PKCα) 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 A₂. PIP₂ did not induce significant changes in secondary structure of the membrane-bound PKCα-C2 domain, nor did binding of the PKCα-C2 domain change the dichroic ratios of the lipid chains, whereas the C2 domain from phospholipase A₂ 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 β-sheets from that of the β-strands within the sheet. When associated with POPC/POPS membranes, the β-sandwich of the PKCα-C2 domain is inclined at an angle α=35° 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 α=44° in the presence of PIP₂, indicating that the β-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 PKCα.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cell Membrane / metabolism*
  • Cytosol / metabolism
  • Humans
  • Models, Molecular
  • Phosphatidylcholines / metabolism*
  • Phosphatidylserines / metabolism*
  • Phospholipases A2 / metabolism*
  • Protein Kinase C-alpha / chemistry*
  • Protein Kinase C-alpha / metabolism*
  • Protein Structure, Tertiary
  • Spectrophotometry, Infrared
  • Spectroscopy, Fourier Transform Infrared

Substances

  • Phosphatidylcholines
  • Phosphatidylserines
  • 1-palmitoyl-2-oleoylglycero-3-phosphoserine
  • Protein Kinase C-alpha
  • Phospholipases A2
  • 1-palmitoyl-2-oleoylphosphatidylcholine