Effect of PIP2 binding on the membrane docking geometry of PKC alpha C2 domain: an EPR site-directed spin-labeling and relaxation study.
Protein kinase C isoform alpha (PKCalpha) is a ubiquitous, conventional PKC enzyme that possesses a conserved C2 domain. Upon activation by cytoplasmic Ca (2+) ions, the C2 domain specifically binds to the plasma membrane inner leaflet where it recognizes the target lipids phosphatidylserine (PS) and phosphatidylinositol-4,5-bisphosphate (PIP 2). The membrane penetration depth and docking angle of the membrane-associated C2 domain is not well understood. The present study employs EPR site-directed spin labeling and relaxation methods to generate a medium-resolution model of the PKCalpha C2 domain docked to a membrane of lipid composition similar to the plasma membrane inner leaflet. The approach measures EPR depth parameters for 10 function-retaining spin labels coupled to the C2 domain, and for spin labels coupled to depth calibration molecules. The resulting depth parameters, together with the known structure of the free C2 domain, provide a sufficient number of constraints to define two membrane docking geometries for C2 domain bound to physiological membranes lacking or containing PIP 2, respectively. In both the absence and presence of PIP 2, the two bound Ca (2+) ions of the C2 domain lie near the anionic phosphate plane in the headgroup region, consistent with the known ability of the Ca (2+) and membrane-binding loops (CMBLs) to bind the headgroup of the PS target lipid. In the absence of PIP 2, the polybasic lipid binding site on the beta3-beta4 hairpin is occupied with PS, but in the presence of PIP 2 this larger, higher affinity target lipid competitively displaces PS and causes the long axis of the domain to tilt 40 +/- 10 degrees toward the bilayer normal. The ability of the beta3-beta4 hairpin site to bind PS as well as PIP 2 extends the lifetime of the membrane-docked state and is predicted to enhance the kinase turnover number of PKCalpha during a single membrane docking event. In principle, PIP 2-induced tilting of the C2 domain could modulate the activity of membrane-docked PKCalpha as it diffuses between membrane regions with different local PS and PIP 2 concentrations. Finally, the results demonstrate that EPR relaxation methods are sufficiently sensitive to detect signaling-induced changes in the membrane docking geometries of peripheral membrane proteins.
Landgraf
Kyle E
KE
Department of Chemistry and Biochemistry and the Molecular Biophysics Program, University of Colorado, Boulder, Colorado 80309-0215, USA.
Malmberg
Nathan J
NJ
Falke
Joseph J
JJ
D000818:Animals; D002417:Cattle; D016503:Drug Delivery Systems; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods; D006801:Humans; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding / Q000235:genetics; D051571:Protein Kinase C-alpha / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D013113:Spin Labels; D013552:Swine
D007527:Isoenzymes; D008565:Membrane Proteins; D019269:Phosphatidylinositol 4,5-Diphosphate; D013113:Spin Labels; D051571:Protein Kinase C-alpha