PGI & TXA SYNTHASES-MEMBRANE ANCHOR STRUCTURE/FUNCTION

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DESCRIPTION (Adapted from Abstract) The long-term goal of this project is to understand how the native, membrane-bound structures of two eicosanoid-synthesizing cytochrome P450s, thromboxane A2 synthase (TXAS) and prostaglandin I2 synthase (PGIS), influence enzyme function and coordination with prostaglandin H2 synthase (PGHS), and to understand the membrane topology of mammalian P450 superfamily. TXAS converts prostaglandin H2 (PGH2), produced by PGHS in endoplasmic reticulum. (ER) lumen to thromboxane A2 (TXA2) on the cytoplasmic side of the ER. TXA2 is a mediator with potent platelet aggregatory and vasoconstrictive properties. PGIS converts the same substrate, PGH2, to prostaglandin I2 (PGI2), with biological activities that are opposite to TXA2. TXA2 and PGI2, play important roles in a wide variety of physiological and pathological processes affecting blood and vasculature. Biosynthesis of TXA2 or PGI2 involves coordination of either TXAS or PGIS with PGHS anchored on the opposite side of the ER membrane. This raises the possibility that the membrane anchors influence the coordination. PI's research during past funding indicates that the large cytoplasmic domain of PGIS is anchored to the ER membrane by a single N-terminal anchor segment similar to that in other microsomal P450s, but different from TXAS, which appears to have two membrane anchor segments. The results also indicate that the PGIS N-terminal membrane anchor is near the opening of the substrate access channel and influences enzyme reaction rate. These results led PI to hypothesize that PGIS and TXAS have specific substrate-recognition sites in their N-terminal membrane domains, which facilitate substrate access to their active site channels. PI also suspects that the helix F/G loop of TXAS and PGIS contains a membrane contact region distinct from the N-termini. Crystallographic studies suggest that the catalytic domains of PGHS are anchored to the ER lumen by helices A-D, and thus directly abutting the substrate channel to the ER membrane. To test these hypotheses, Dr. Ruan proposes to analyze and compare the membrane anchor domains of TXAS, PGIS, PGHS and P450 2CI, by a variety of techniques, including immunocytochemistry with domain-specific antibodies, molecular modeling, circular dichroism and NMR. Complete 3D structures of PGIS, TXAS and P450 2C I N-terminal membrane segments will be obtained to provide the solution structures in the membrane environment, complementing existing crystallographic data for P450. The Specific Aims are to: 1) Characterize TXAS and PGIS N-terminal membrane anchor domain which influence the enzyme catalysis, localize the residues important to function and determine the 3D structures of the complex with the interactions between the substrate analog and the membrane domains; 2) Identify membrane contact regions in helix F/G loops of TXAS and PGIS and further define their topology and substrate access channels with respect to the ER membrane; 3) Determine the 3D structure of a synthetic peptide mimicking P450 2C1 N-terminal membrane segment to build a general topology and 3D structural models for microsomal P450s; 4) Determine membrane topology and 3D-solution structure of membrane anchor domains of PGHS-1 and -2 in membrane environment. These studies will provide new insight into how the movement of hydrophobic substrates from membrane compartment to enzyme active sites and between the active sites in case of PGHS/PGIS and PGHS/TXAS is accomplished in an efficient manner within the membrane environment, which complement P450 crystallographic data.