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Characterization of the secondary structure and membrane interaction of the putative membrane anchor domains of prostaglandin I2 synthase and cytochrome P450 2C1.

Prostaglandin I2 synthase (PGIS) produces prostaglandin I2 (PGI2) which has opposite actions on platelet aggregatory and vasoconstrictive properties compared to thromboxane A2 (TXA2) produced from the same substrate by another P450 enzyme, thromboxane A2 synthase (TXAS). PGIS and TXAS have only 16% amino acid sequence identity. Hydropathy analysis suggests that the putative NH2-terminal membrane anchor domain of PGIS is similar to many other membrane-bound microsomal P450s, which are believed to be anchored by a single transmembrane segment, and thus different from the TXAS anchor, which appears to have two transmembrane segments. To characterize the membrane anchor function of the PGIS NH2-terminal region, we have used the peptidoliposome reconstitution assay to identify the membrane anchor segment in the PGIS NH2-terminal domain and compared it with the anchor segment of P450 2C1. Four peptides, mimicking putative NH2-terminal membrane anchor segments of PGIS and P450 2C1, containing residues 1-28 (PGIS-LP1 and P450 2C1-LP1) or residues 25-54 (PGIS-LP2 and P450 2C1-LP2), were synthesized and their ability to insert in a lipid bilayer was evaluated. The results indicated that both LP1 peptides of PGIS and P450 2C1 became bound to the lipid bilayer, whereas both LP2 peptides did not bind the lipid. The two LP1 peptides were further characterized as to their conformation using CD spectroscopy. Helical structure induced in these peptides by addition of trifluoroethanol, dodecylphosphocholine, or incorporation into liposomes indicated that these segments tend to adopt a helical structure in a hydrophobic environment and thus could function as membrane anchor segments. These results support the hypothesis that PGIS and TXAS interact with the endoplasmic reticulum membrane in different ways, in which the NH2-terminal anchor domain of PGIS, as with P450 2C1, appears to have a single transmembrane segment.