Topography of the Prostaglandin Endoperoxide H2 Synthase-2 in Membranes

Zahra Mirafzali,Jeffrey R Leipprandt,John L Mccracken,David L Dewitt

doi:10.1074/jbc.m605206200

Zahra Mirafzali, Jeffrey R Leipprandt + Show 2 more

Open Access

https://doi.org/10.1074/jbc.m605206200

Copy DOI

Abstract

The topology of association of the monotopic protein cyclooxygenase-2 (COX-2) with membranes has been examined using EPR spectroscopy of spin-labeled recombinant human COX-2. Twenty-four mutants, each containing a single free cysteine substituted for an amino acid in the COX-2 membrane binding domain were expressed using the baculovirus system and purified, then conjugated with a nitroxide spin label and reconstituted into liposomes. Determining the relative accessibility of the nitroxide-tagged amino acid side chains for the solubilized COX-2 mutants, or COX-2 reconstituted into liposomes to nonpolar (oxygen) and polar (NiEDDA or CrOx) paramagnetic reagents allowed us to map the topology of COX-2 interaction with the lipid bilayer. When spin-labeled COX-2 was reconstituted into liposomes, EPR power saturation curves showed that side chains for all but two of the 24 mutants tested had limited accessibility to both polar and nonpolar paramagnetic relaxation agents, indicating that COX-2 associates primarily with the interfacial membrane region near the glycerol backbone and phospholipid head groups. Two amino acids, Phe(66) and Leu(67), were readily accessible to the non-polar relaxation agent oxygen, and thus likely inserted into the hydrophobic core of the lipid bilayer. However these residues are co-linear with amino acids in the interfacial region, so their extension into the hydrophobic core must be relatively shallow. EPR and structural data suggest that membrane interaction of COX-2 is also aided by partitioning of 4 aromatic amino acids, Phe(59), Phe(66), Tyr(76), and Phe(84) to the interfacial region, and by the electrostatic interactions of two basic amino acids, Arg(62) and Lys(64), with the phospholipid head groups.

Highlights

Essential and regulatory role in prostaglandin synthesis and because they are the site of action of non-steroidal anti-inflammatory drugs (NSAIDs) such as aspirin, Ibuprofen, Naproxin, Vioxx௡, and Celebrex௡
Site-directed spin labeling requires that a protein contain only a single reactive cysteine; a cysteine-less COX-2 template was prepared by mutating to serines the two cysteines (229 and 526) that do not form disulfide bonds [9]
Plot of ⌬H0Ϫ1 calculated from the spectra of the nitroxide-la- These experiments show that only two amino acids of beled COX-2 mutants (Fig. 4)

Summary

EXPERIMENTAL PROCEDURES

Materials—1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 1,2-dioleoyl-sn-glycero-3-[phospho-L-serine] (Sodium Salt) (DOPS) were obtained from Avanti Polar Lipids Inc. (Alabaster, AL). The resulting film was hydrated in a buffer of 25 mM Tris-Cl, pH 7.4 containing 50 mM KCl for 1 h at a concentration of 38 mM, vortexed thoroughly and extruded 20 times through a 0.1-␮m pore diameter polycarbonate filter using a Mini-Extruder (Avanti Polar Lipids). This process produces a transparent milky suspension. Incorporation of COX into the Preformed Liposomes—Purified COX (20 –30 nmol, 20 mg/ml) in 25 mM Tris-Cl, pH 7.4 containing 50 mM KCl was added to the preformed liposomes at a protein:phospholipid molar ratios of 1:500 in a total volume of 0.5 ml and incubated for 30 min at 37 °C [7].

RESULTS

Relative activity after mutant conjugation with MTSSLa conjugation with MTSSL

Helix C

DISCUSSION