Topography of the prostaglandin endoperoxide H2 synthase-2 in membranes.
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.
MirAfzali
Zahra
Z
Encapsula NanoSciences, Nashville, Tennessee 37203, USA.
Leipprandt
Jeffrey R
JR
McCracken
John L
JL
DeWitt
David L
DL
D000818:Animals; D002384:Catalysis; D051546:Cyclooxygenase 2 / Q000737:chemistry*; D004578:Electron Spin Resonance Spectroscopy; D006801:Humans; D008081:Liposomes / Q000737:chemistry; D017433:Protein Structure, Secondary; D018411:Spodoptera; D055672:Static Electricity
D008081:Liposomes; D051546:Cyclooxygenase 2