Functional Investigations of Thromboxane Synthase (CYP5A1) in Lipid Bilayers of Nanodiscs

Abstract

CYP5A1 is a membrane-associated cytochrome P450 that metabolizes the cyclooxygenase product prostaglandin (PGH2 ) into thromboxane A2 (TXA2 ), a potent inducer of vasoconstriction and platelet aggregation. Although CYP5A1 is an ER-bound protein, the role of membranes in modulating the thermodynamics and kinetics of substrate binding to this protein has not been elucidated. In this work, we incorporated thromboxane synthase into lipid bilayers of nanodiscs for functional studies. We measured the redox potential of CYP5A1 in nanodiscs and showed that the redox potential is within a similar range of other drug-metabolizing P450 enzymes in membranes. Further, we showed that binding of substrate to CYP5A1 can induce conformational changes in the protein that block small-molecule ligand egress by measuring the kinetics of cyanide binding to CYP5A1 as a function of substrate concentration. Notably, we observed that sensitivity to cyanide binding was different for two substrate analogues, U44069 and U46619, thus indicating that they bind differently to the active site of CYP5A1. We also characterized the effects of the different lipids on CYP5A1 catalytic activity by using nanodiscs to create unary, binary, and ternary lipid systems. CYP5A1 activity increased dramatically in the presence of charged lipids POPS and POPE, as compared to the unary POPC system. These results suggest the importance of lipid composition on modulating the activity of CYP5A1 to increase thromboxane formation.

Incorporation of charged residues in the CYP2J2 F-G loop disrupts CYP2J2–lipid bilayer interactions

“…While seminal studies correctly identified the general nature of the protein-membrane binding and topology (5), more recent examples have elucidated the interactions at the molecular and atomistic levels (6-8). Notably, P450 interactions with membranes have been shown to regulate ligand binding mechanisms (9), membrane depth of insertion (10), redox potentials (11), enzyme stability (12), structure and orientation (6). Additionally, advanced computational approaches have revealed the dynamic nature of CYPs and how their hydrophobic motifs interact with the membrane bilayer (6, 8).…”

Endocannabinoids Anandamide and 2-Arachidonoylglycerol Are Substrates for Human CYP2J2 Epoxygenase

“…For all studies with recombinant protein, we used the nanoscale lipid bilayers of nanodiscs (ND) to solubilize and stabilize membrane-bound CYP2J2. These model membranes have been previously used to functionally stabilize membrane protein in solution and on surfaces (Nath et al, 2007;Bayburt and Sligar, 2010;Denisov and Sligar, 2011;Das et al, 2014;Orlando et al, 2014). Importantly, the use of these model membranes significantly reduced scattering and enabled spectroscopic characterization by increasing the solubility of the hydrophobic lipid substrates and membrane proteins.…”

Development of Poly Unsaturated Fatty Acid Derivatives of Aspirin for Inhibition of Platelet Function

“…5A). We previously showed that TXAS facilitates robust biophysical studies in nanodiscs compared with naked protein in detergent solution (Das et al, 2014).…”

“…Thromboxane synthase (TXAS) was expressed and purified as previously mentioned (Das et al, 2014). The gene for TXAS was obtained from OriGene (Rockville, MD) and modified at the N terminus for expression in Escherichia coli as described.…”

“…Amberlite was added to remove detergents and initiate the formation of nanodiscs. The TXAS nanodiscs were then purified using size exclusion chromatography as previously mentioned (Das et al, 2014).…”