NDLI: Effect of PIP2 Binding on the Membrane Docking Geometry of PKCα C2 Domain: An EPR Site-Directed Spin-Labeling and Relaxation Study †

Content Provider	PubMed Central
Author	Landgraf, Kyle E. Malmberg, Nathan J. Falke, Joseph J.
Copyright Year	2008
Abstract	Protein kinase C isoform alpha (PKCα) is a ubiquitous, conventional PKC enzyme that possesses a conserved C2 domain. Upon activation by cytoplasmic Ca2+ 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 (PIP2). 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 PKCα 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 PIP2, respectively. In both the absence and presence of PIP2, the two bound Ca2+ ions of the C2 domain lie near the anionic phosphate plane in the headgroup region, consistent with the known ability of the Ca2+ and membrane-binding loops (CMBLs) to bind the headgroup of the PS target lipid. In the absence of PIP2, the polybasic lipid binding site on the β3-β4 hairpin is occupied with PS, but in the presence of PIP2 this larger, higher affinity target lipid competitively displaces PS and causes the long axis of the domain to tilt 40 ± 10° toward the bilayer normal. The ability of the β3-β4 hairpin site to bind PS as well as PIP2 extends the lifetime of the membrane-docked state and is predicted to enhance the kinase turnover number of PKCα during a single membrane docking event. In principle, PIP2-induced tilting of the C2 domain could modulate the activity of membrane-docked PKCα as it diffuses between membrane regions with different local PS and PIP2 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.
Related Links	http://dx.doi.org/10.1021/bi800711t
Ending Page	8316
Page Count	16
Starting Page	8301
File Format	PDF
ISSN	15204995
e-ISSN	15204995
Journal	Biochemistry
Issue Number	32
Volume Number	47
Language	English
Publisher Date	2008-08-12
Access Restriction	Open
Subject Keyword	Research in Higher Education
Content Type	Text
Resource Type	Article
Subject	Biochemistry

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