Membrane docking geometry and target lipid stoichiometry of membrane-bound PKCα C2 domain: A combined molecular dynamics and experimental study

Chun Liang Lai, Kyle E. Landgraf, Gregory A. Voth, Joseph J. Falke

Research output: Contribution to journalArticle

38 Scopus citations

Abstract

Protein kinase Cα (PKCα) possesses a conserved C2 domain (PKCα C2 domain) that acts as a Ca2+-regulated membrane targeting element. Upon activation by Ca2+, the PKCα C2 domain directs the kinase protein to the plasma membrane, thereby stimulating an array of cellular pathways. At sufficiently high Ca2+ concentrations, binding of the C2 domain to the target lipid phosphatidylserine (PS) is sufficient to drive membrane association; however, at typical physiological Ca2+ concentrations, binding to both PS and phosphoinositidyl-4,5-bisphosphate (PIP2) is required for specific plasma membrane targeting. Recent EPR studies have revealed the membrane docking geometries of the PKCα C2 domain docked to (i) PS alone and (ii) both PS and PIP2 simultaneously. These two EPR docking geometries exhibit significantly different tilt angles relative to the plane of the membrane, presumably induced by the large size of the PIP2 headgroup. The present study utilizes the two EPR docking geometries as starting points for molecular dynamics simulations that investigate atomic features of the protein-membrane interaction. The simulations yield approximately the same PIP2-triggered change in tilt angle observed by EPR. Moreover, the simulations predict a PIP2:C2 stoichiometry approaching 2:1 at a high PIP2 mole density. Direct binding measurements titrating the C2 domain with PIP2 in lipid bilayers yield a 1:1 stoichiometry at moderate mole densities and a saturating 2:1 stoichiometry at high PIP2 mole densities. Thus, the experiment confirms the target lipid stoichiometry predicted by EPR-guided molecular dynamics simulations. Potential biological implications of the observed docking geometries and PIP2 stoichiometries are discussed.

Original languageEnglish (US)
Pages (from-to)301-310
Number of pages10
JournalJournal of Molecular Biology
Volume402
Issue number2
DOIs
StatePublished - Sep 2010

Keywords

  • Conserved membrane targeting domain
  • Molecular dynamics
  • Phosphatidylinositol-4,5-bisphosphate regulation
  • Positive cooperativity
  • Protein kinase C activation

ASJC Scopus subject areas

  • Structural Biology
  • Molecular Biology

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