TY - JOUR
T1 - Membrane docking geometry and target lipid stoichiometry of membrane-bound PKCα C2 domain
T2 - A combined molecular dynamics and experimental study
AU - Lai, Chun Liang
AU - Landgraf, Kyle E.
AU - Voth, Gregory A.
AU - Falke, Joseph J.
N1 - Funding Information:
This study was supported by grants from the National Institutes of Health ( R01-GM063796 to G.A.V., R01-GM063235 to J.J.F., and T32-GM065103 to K.E.L.). Computational resources were provided by the National Science Foundation through the TeraGrid computing resources of the Texas Advanced Computing Center, the Pittsburgh Supercomputing Center, the San Diego Supercomputing Center, and the National Institute for Computational Sciences. The authors thank Dr. Gary Ayton for his valuable input. The contribution of C.-L.L. and G.A.V. to this work was initiated at the Center for Biophysical Modeling and Simulation, University of Utah.
PY - 2010/9
Y1 - 2010/9
N2 - 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.
AB - 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.
KW - Conserved membrane targeting domain
KW - Molecular dynamics
KW - Phosphatidylinositol-4,5-bisphosphate regulation
KW - Positive cooperativity
KW - Protein kinase C activation
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U2 - 10.1016/j.jmb.2010.07.037
DO - 10.1016/j.jmb.2010.07.037
M3 - Article
C2 - 20659476
AN - SCOPUS:77956789005
SN - 0022-2836
VL - 402
SP - 301
EP - 310
JO - Journal of Molecular Biology
JF - Journal of Molecular Biology
IS - 2
ER -