Homotropic cooperativity of monomeric cytochrome P450 3A4 in a nanoscale native bilayer environment

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Abstract

Mechanistic studies of mammalian cytochrome P450s are often obscured by the phase heterogeneity of solubilized preparations of membrane enzymes. The various protein–protein aggregation states of microsomes, detergent solubilized cytochrome or a family of aqueous multimeric complexes can effect measured substrate binding events as well as subsequent steps in the reaction cycle. In addition, these P450 monooxygenases are normally found in a membrane environment and the bilayer composition and dynamics can also effect these catalytic steps. Here, we describe the structural and functional characterization of a homogeneous monomeric population of cytochrome P450 3A4 (CYP 3A4) in a soluble nanoscale membrane bilayer, or Nanodisc [Nano Lett. 2 (2002) 853]. Cytochrome P450 3A4:Nanodisc assemblies were formed and purified to yield a 1:1 ratio of CYP 3A4 to Nanodisc. Solution small angle X-ray scattering was used to structurally characterize this monomeric CYP 3A4 in the membrane bilayer. The purified CYP 3A4:Nanodiscs showed a heretofore undescribed high level of homotropic cooperativity in the binding of testosterone. Soluble CYP 3A4:Nanodisc retains its known function and shows prototypic hydroxylation of testosterone when driven by hydrogen peroxide. This represents the first functional characterization of a true monomeric preparation of cytochrome P450 monooxygenase in a phospholipid bilayer and elucidates new properties of the monomeric form.

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Chemicals

Imidazole, sodium cholate, testosterone, 6β-OH testosterone, and androstenedione were purchased from Sigma Chemical (St. Louis, MO), Emulgen 913 was from Karlan Research Products (Santa Rosa, CA), palmitoyl-oleoyl-phosphatidylcholine (POPC) was from Avanti Polar Lipids (Alabaster, AL), and hydrogen peroxide was from Fisher Chemical (Pittsburgh, PA).

Cytochrome P450 3A4 expression and purification

Cytochrome P450 3A4 was expressed from the NF-14 construct in the PCWori+ vector with a C-terminal pentahistidine tag generously provided by Dr. F.

Results

To guarantee monomeric cytochrome P450 3A4 in a native like phospholipid bilayer environment, we utilized Nanodisc technology. This methodology uses a MSP which self-assembles phospholipid and a membrane protein target into soluble discoidal bilayers ∼10 nm in diameter. The length of the encircling “belt” defines the size of the solubilized bilayer domain and hence can control the oligomerization state of incorporated targets. Self-assembly of cytochrome P450 3A4 was as described in Experimental

Discussion

Human cytochrome P450 3A4 was self-assembled into soluble Nanodiscs, in a clean 1:1 complex. These CYP 3A4:Nanodiscs allow the study of individual cytochrome P450 molecules in a system which mimics the native biological membrane, and is free of artifacts caused by aggregation. SAXS shows that these complexes have a monomeric CYP 3A4 molecule in a nanoscale phospholipid bilayer environment. Analysis of the possible models of CYP3A4 incorporated into the Nanodisc bilayer as illustrated at the

Acknowledgments

We gratefully acknowledge Dr. F.P. Guengerich for the clone of cytochrome P450 3A4. We also thank Aretta Weber for her excellent editorial help as well as a special thanks to Dr. Timothy Bayburt and Yelena Grinkova for their helpful discussion and expertise. Portions of this work were performed at the DuPont-Northwestern-Dow Collaborative Access Team (DND-CAT) Synchrotron Research Center located at Sector 5 of the Advanced Photon Source. DND-CAT is supported by the E.I. DuPont de Nemours & Co.,

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