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Interfacial membrane docking of cytosolic phospholipase A2 C2 domain using electrostatic potential-modulated spin relaxation magnetic resonance.

ABSTRACT: The C2 domain of cytosolic phospholipase A2 (C2cPLA2) plays an important role in calcium-dependent transfer of the protein from the cytosol to internal cellular membranes as a prelude for arachidonate release from membrane phospholipids. By using a recently developed electron paramagnetic resonance approach together with 13 site-specifically nitroxide spin labeled C2cPLA2s and membrane-permeant and -impermeant spin relaxants, we have determined the orientation of C2cPLA2 with respect to the surface of vesicles of the phospholipid 1,2-dioleoyl-sn-glycero-3-phosphomethanol. The structure reveals that the two calcium-binding regions on C2cPLA2 that display hydrophobic residues, CBR1 and CBR3, are partially inserted into the core of the membrane. CBR2 that contains predominantly hydrophilic residues is close to the membrane but not inserted. The long axis of the cylindrical C2cPLA2 molecule is tilted with respect to the bilayer normal, which brings a cluster of basic protein residues close to the phospholipid headgroups. Such an orientation places the two bound calcium ions close to the membrane surface. All together, the results provide structural support for previous proposals that binding of C2cPLA2 to the membrane interface is driven in part by insertion of hydrophobic surface loops into the membrane core. The results are contrasted with previous studies of the interfacial binding of the first C2 domain of synaptotagmin I, which has shorter surface loops that display basic residues for electrostatic interaction with the bilayer surface.

SUBMITTER: Ball A

PROVIDER: S-EPMC21967 | biostudies-literature | 1999 Jun

REPOSITORIES: biostudies-literature

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Interfacial membrane docking of cytosolic phospholipase A2 C2 domain using electrostatic potential-modulated spin relaxation magnetic resonance.

Ball A A Nielsen R R Gelb M H MH Robinson B H BH

Proceedings of the National Academy of Sciences of the United States of America 19990601 12

The C2 domain of cytosolic phospholipase A2 (C2cPLA2) plays an important role in calcium-dependent transfer of the protein from the cytosol to internal cellular membranes as a prelude for arachidonate release from membrane phospholipids. By using a recently developed electron paramagnetic resonance approach together with 13 site-specifically nitroxide spin labeled C2cPLA2s and membrane-permeant and -impermeant spin relaxants, we have determined the orientation of C2cPLA2 with respect to the surf ...[more]

PMID: 10359764

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Project description: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.

Dataset Information

Interfacial membrane docking of cytosolic phospholipase A2 C2 domain using electrostatic potential-modulated spin relaxation magnetic resonance.

Publications

Interfacial membrane docking of cytosolic phospholipase A2 C2 domain using electrostatic potential-modulated spin relaxation magnetic resonance.

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