Dataset Information

Cyclooxygenase-2 catalysis and inhibition in lipid bilayer nanodiscs.

ABSTRACT: Cyclooxygenases (COX-1 and COX-2) oxygenate arachidonic acid (AA) to generate prostaglandins. The enzymes associate with one leaflet of the membrane bilayer. We utilized nanodisc technology to investigate the function of human (hu) COX-2 and murine (mu) COX-2 in a lipid bilayer environment. huCOX-2 and muCOX-2 were incorporated into nanodiscs composed of POPC, POPS, DOPC, or DOPS phospholipids. Size-exclusion chromatography and negative stain electron microscopy confirm that a single COX-2 homodimer is incorporated into the nanodisc scaffold. Nanodisc-reconstituted COX-2 exhibited similar kinetic profiles for the oxygenation of AA, eicosapentaenoic acid, and 1-arachidonoyl glycerol compared to those derived using detergent solubilized enzyme. Moreover, changing the phospholipid composition of the nanodisc did not alter the ability of COX-2 to oxygenate AA or to be inhibited by various nonselective NSAIDs or celecoxib. The cyclooxygenase activity of nanodisc-reconstituted COX-2 was reduced by aspirin acetylation and potentiated by the nonsubstrate fatty acid palmitic acid to the same extent as detergent solubilized enzyme, independent of phospholipid composition. The stabilization and maintenance of activity afforded by the incorporation of the enzyme into nanodiscs generates a native-like lipid bilayer environment to pursue studies of COX utilizing solution-based techniques that are otherwise not tractable in the presence of detergents.

SUBMITTER: Orlando BJ

PROVIDER: S-EPMC3967765 | biostudies-literature | 2014 Mar

REPOSITORIES: biostudies-literature

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Cyclooxygenase-2 catalysis and inhibition in lipid bilayer nanodiscs.

Orlando Benjamin J BJ McDougle Daniel R DR Lucido Michael J MJ Eng Edward T ET Graham Leigh Ann LA Schneider Claus C Stokes David L DL Das Aditi A Malkowski Michael G MG

Archives of biochemistry and biophysics 20140203

Cyclooxygenases (COX-1 and COX-2) oxygenate arachidonic acid (AA) to generate prostaglandins. The enzymes associate with one leaflet of the membrane bilayer. We utilized nanodisc technology to investigate the function of human (hu) COX-2 and murine (mu) COX-2 in a lipid bilayer environment. huCOX-2 and muCOX-2 were incorporated into nanodiscs composed of POPC, POPS, DOPC, or DOPS phospholipids. Size-exclusion chromatography and negative stain electron microscopy confirm that a single COX-2 homod ...[more]

PMID: 24503478

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Project description:Lipid nanodiscs are small synthetic lipid bilayer structures that are stabilized in solution by special circumscribing (or scaffolding) proteins or polymers. Because they create native-like environments for transmembrane proteins, lipid nanodiscs have become a powerful tool for structural determination of this class of systems when combined with cryo-electron microscopy or nuclear magnetic resonance. The elastic properties of lipid bilayers determine how the lipid environment responds to membrane protein perturbations, and how the lipid in turn modifies the conformational state of the embedded protein. However, despite the abundant use of nanodiscs in determining membrane protein structure, the elastic material properties of even pure lipid nanodiscs (i.e., without embedded proteins) have not yet been quantitatively investigated. A major hurdle is due to the inherently nonlocal treatment of the elastic properties of lipid systems implemented by most existing methods, both experimental and computational. In addition, these methods are best suited for very large "infinite" size lipidic assemblies, or ones that contain periodicity, in the case of simulations. We have previously described a computational analysis of molecular dynamics simulations designed to overcome these limitations, so it allows quantification of the bending rigidity (KC) and tilt modulus (κt) on a local scale even for finite, nonperiodic systems, such as lipid nanodiscs. Here we use this computational approach to extract values of KC and κt for a set of lipid nanodisc systems that vary in size and lipid composition. We find that the material properties of lipid nanodiscs are different from those of infinite bilayers of corresponding lipid composition, highlighting the effect of nanodisc confinement. Nanodiscs tend to show higher stiffness than their corresponding macroscopic bilayers, and moreover, their material properties vary spatially within them. For small-size MSP1 nanodiscs, the stiffness decreases radially, from a value that is larger in their center than the moduli of the corresponding bilayers by a factor of ∼2-3. The larger nanodiscs (MSP1E3D1 and MSP2N2) show milder spatial changes of moduli that are composition dependent and can be maximal in the center or at some distance from it. These trends in moduli correlate with spatially varying structural properties, including the area per lipid and the nanodisc thickness. Finally, as has previously been reported, nanodiscs tend to show deformations from perfectly flat circular geometries to varying degrees, depending on size and lipid composition. The modulations of lipid elastic properties that we find should be carefully considered when making structural and functional inferences concerning embedded proteins.

Dataset Information

Cyclooxygenase-2 catalysis and inhibition in lipid bilayer nanodiscs.

Publications

Cyclooxygenase-2 catalysis and inhibition in lipid bilayer nanodiscs.

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