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Effects of Membrane Mimetics on Cytochrome P450-Cytochrome b5 Interactions Characterized by NMR Spectroscopy*

  • Meng Zhang
    Affiliations
    From the Department of Chemistry and Biophysics, University of Michigan, Ann Arbor, Michigan 48109-1055 and
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  • Rui Huang
    Affiliations
    From the Department of Chemistry and Biophysics, University of Michigan, Ann Arbor, Michigan 48109-1055 and
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  • Sang-Choul Im
    Affiliations
    the Department of Anesthesiology, University of Michigan and Veterans Affairs Medical Center, Ann Arbor, Michigan 48105
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  • Lucy Waskell
    Affiliations
    the Department of Anesthesiology, University of Michigan and Veterans Affairs Medical Center, Ann Arbor, Michigan 48105
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  • Ayyalusamy Ramamoorthy
    Correspondence
    To whom the correspondence should be addressed: Dept. of Chemistry and Biophysics, University of Michigan, Ann Arbor, MI 48109-1055. Tel.: 734-647-6572; Fax: 734-764-3323;
    Affiliations
    From the Department of Chemistry and Biophysics, University of Michigan, Ann Arbor, Michigan 48109-1055 and
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  • Author Footnotes
    * This work was supported, in whole or in part, by National Institutes of Health Grants GM084018 and GM095640 (to A. R.) and GM35533 (to L. W.). This work was also supported by a VA Merit grant (to L. W.).
Open AccessPublished:March 20, 2015DOI:https://doi.org/10.1074/jbc.M114.597096
      Mammalian cytochrome P450 (P450) is a membrane-bound monooxygenase whose catalytic activities require two electrons to be sequentially delivered from its redox partners: cytochrome b5 (cytb5) and cytochrome P450 reductase, both of which are membrane proteins. Although P450 functional activities are known to be affected by lipids, experimental evidence to reveal the effect of membrane on P450-cytb5 interactions is still lacking. Here, we present evidence for the influence of phospholipid bilayers on complex formation between rabbit P450 2B4 (CYP2B4) and rabbit cytb5 at the atomic level, utilizing NMR techniques. General line broadening and modest chemical shift perturbations of cytb5 resonances characterize CYP2B4-cytb5 interactions on the intermediate time scale. More significant intensity attenuation and a more specific protein-protein binding interface are observed in bicelles as compared with lipid-free solution, highlighting the importance of the lipid bilayer in stabilizing stronger and more specific interactions between CYP2B4 and cytb5, which may lead to a more efficient electron transfer. Similar results observed for the interactions between CYP2B4 lacking the transmembrane domain (tr-CYP2B4) and cytb5 imply interactions between tr-CYP2B4 and the membrane surface, which might assist in CYP2B4-cytb5 complex formation by orienting tr-CYP2B4 for efficient contact with cytb5. Furthermore, the observation of weak and nonspecific interactions between CYP2B4 and cytb5 in micelles suggests that lipid bilayer structures and low curvature membrane surface are preferable for CYP2B4-cytb5 complex formation. Results presented in this study provide structural insights into the mechanism behind the important role that the lipid bilayer plays in the interactions between P450s and their redox partners.

      Introduction

      Cytochrome P450 (P450)
      The abbreviations used are:
      P450 and P420
      cytochrome P450 and P420, respectively
      tr-P450
      truncated cytochrome P450 (cytochrome P450 lacking the N-terminal transmembrane domain)
      cytb5
      cytochrome b5
      CYP2B4
      cytochrome P450 2B4
      TM
      transmembrane
      CPR
      cytochrome P450 reductase
      wt- and tr-CYP2B4
      full-length wild-type and truncated cytochrome P450 2B4, respectively
      ER
      endoplasmic reticulum
      DLPC
      1,2-dilauroyl-sn-glycero-3-phosphocholine
      DHPC
      1,2-dihexanoyl-sn-glycero-3-phosphocholine
      DPC
      n-dodecylphosphocholine
      TROSY-HSQC
      transverse relaxation optimized spectroscopy-heteronuclear single quantum correlation
      CSP
      chemical shift perturbation.
      monooxygenases are a ubiquitous superfamily of enzymes found in all living kingdoms, including plants, animals, bacteria, and fungi (
      • Dürr U.H.N.
      • Waskell L.
      • Ramamoorthy A.
      The cytochromes P450 and b5 and their reductases: promising targets for structural studies by advanced solid-state NMR spectroscopy.
      ). Eukaryotic P450s are membrane-bound proteins, usually containing a large soluble domain and a single α-helical transmembrane (TM) domain (
      • Danielson P.B.
      The cytochrome P450 superfamily: biochemistry, evolution and drug metabolism in humans.
      ). A total of 57 human P450s have been discovered (
      • Guengerich F.P.
      • Wu Z.-L.
      • Bartleson C.J.
      Function of human cytochrome P450s: characterization of the orphans.
      ,
      • Nelson D.R.
      Comparison of P450s from human and fugu: 420 million years of vertebrate P450 evolution.
      ) and are responsible for the metabolism of a wide range of endogenous and exogenous substrates, including sterols, vitamins, fatty acids, environmental pollutants, and over 50% of marketed drugs (
      • Dürr U.H.N.
      • Waskell L.
      • Ramamoorthy A.
      The cytochromes P450 and b5 and their reductases: promising targets for structural studies by advanced solid-state NMR spectroscopy.
      ,
      • Guengerich F.P.
      • Wu Z.-L.
      • Bartleson C.J.
      Function of human cytochrome P450s: characterization of the orphans.
      ,
      • Nebert D.W.
      • Russell D.W.
      Clinical importance of the cytochromes P450.
      ). One of the most studied functions of P450s is the insertion of a single hydroxyl group into hydrophobic compounds, rendering them more hydrophilic for easier excretion from the kidneys (
      • Ortiz de Montellano P.R.
      Hydrocarbon hydroxylation by cytochrome P450 enzymes.
      ). Completion of the hydroxylation reaction requires two electrons to be sequentially delivered to P450, with the first one coming from cytochrome P450 reductase (CPR) and the second one from either CPR or cytochrome b5 (cytb5) (
      • Zhang H.
      • Hamdane D.
      • Im S.-C.
      • Waskell L.
      Cytochrome b5 inhibits electron transfer from NADPH-cytochrome P450 reductase to ferric cytochrome P450 2B4.
      ,
      • Guengerich F.P.
      Cytochrome P450s and other enzymes in drug metabolism and toxicity.
      • Gruenke L.D.
      • Konopka K.
      • Cadieu M.
      • Waskell L.
      The stoichiometry of the cytochrome P-450-catalyzed metabolism of methoxyflurane and benzphetamine in the presence and absence of cytochrome b5.
      ).
      Mammalian P450s and their redox partners (CPR and cytb5) are membrane-bound proteins primarily located on the cytoplasmic side of the endoplasmic reticulum (ER) of hepatic cells (
      • Guengerich F.P.
      Cytochromes P450, drugs, and diseases.
      ). The structure of most mammalian P450s is composed of a large soluble domain and a single α-helical TM domain; cytb5 contains a soluble domain, a single α-helical TM domain, and a linker connecting the aforementioned two domains (
      • Dürr U.H.N.
      • Waskell L.
      • Ramamoorthy A.
      The cytochromes P450 and b5 and their reductases: promising targets for structural studies by advanced solid-state NMR spectroscopy.
      ,
      • Ahuja S.
      • Jahr N.
      • Im S.-C.
      • Vivekanandan S.
      • Popovych N.
      • Le Clair S.V.
      • Huang R.
      • Soong R.
      • Xu J.
      • Yamamoto K.
      • Nanga R.P.
      • Bridges A.
      • Waskell L.
      • Ramamoorthy A.
      A model of the membrane-bound cytochrome b5-cytochrome P450 complex from NMR and mutagenesis data.
      ). It is well documented that the environment provided by the cell membrane, including phospholipids in the ER membrane, is closely tied to the functional activities of many membrane proteins (
      • Coleman R.
      Membrane-bound enzymes and membrane ultrastructure.
      ). Therefore, it is not surprising that phospholipids comprising the ER membrane have been demonstrated to be essential for optimal P450 activities (
      • Lu A.Y.H.
      • Strobel H.W.
      • Coon M.J.
      Hydroxylation of benzphetamine and other drugs by a solubilized form of cytochrome P-450 from liver microsomes: lipid requirement for drug demethylation.
      • Lu A.Y.H.
      • Junk K.W.
      • Coon M.J.
      Resolution of the cytochrome P-450-containing ohgr-hydroxylation system of liver microsomes into three components.
      ,
      • Lu A.Y.H.
      • Strobel H.W.
      • Coon M.J.
      Properties of a solubilized form of the cytochrome P-450-containing mixed-function oxidase of liver microsomes.
      • Strobel H.W.
      • Lu A.Y.H.
      • Heidema J.
      • Coon M.J.
      Phosphatidylcholine requirement in the enzymatic reduction of hemoprotein P-450 and in fatty acid, hydrocarbon, and drug hydroxylation.
      ). It is widely believed that the TM domain of mammalian P450s is not the sole membrane binding segment, but a secondary binding site on the P450 lacking the N-terminal TM domain (tr-P450) exists. A number of different P450s have been reported to bind the membrane in the absence of the TM domain (
      • Pernecky S.J.
      • Larson J.R.
      • Philpot R.M.
      • Coon M.J.
      Expression of truncated forms of liver microsomal P450 cytochromes 2B4 and 2E1 in Escherichia coli: influence of NH2-terminal region on localization in cytosol and membranes.
      • Gillam E.M.
      • Baba T.
      • Kim B.R.
      • Ohmori S.
      • Guengerich F.P.
      Expression of modified human cytochrome P450 3A4 in Escherichia coli and purification and reconstitution of the enzyme.
      ,
      • Sagara Y.
      • Barnes H.J.
      • Waterman M.R.
      Expression in Escherichia coli of functional cytochrome P450c17 lacking its hydrophobic amino-terminal signal anchor.
      ,
      • Larson J.R.
      • Coon M.J.
      • Porter T.D.
      Alcohol-inducible cytochrome P-450IIE1 lacking the hydrophobic NH2-terminal segment retains catalytic activity and is membrane-bound when expressed in Escherichia coli.
      ,
      • Cullin C.
      Two distinct sequences control the targeting and anchoring of the mouse P450 1A1 into the yeast endoplasmic reticulum membrane.
      • Clark B.J.
      • Waterman M.R.
      The hydrophobic amino-terminal sequence of bovine 17α-hydroxylase is required for the expression of a functional hemoprotein in COS 1 cells.
      ). It has been proposed that several loop regions in the tr-P450s interact with the membrane, allowing for a significant portion of the protein surface to be buried inside the membrane (
      • Williams P.A.
      • Cosme J.
      • Sridhar V.
      • Johnson E.F.
      • McRee D.E.
      Mammalian microsomal cytochrome P450 monooxygenase.
      ,
      • Zhao Y.
      • White M.A.
      • Muralidhara B.K.
      • Sun L.
      • Halpert J.R.
      • Stout C.D.
      Structure of microsomal cytochrome P450 2B4 complexed with the antifungal drug bifonazole: insight into P450 conformational plasticity and membrane interaction.
      ); this interaction could further serve in assisting access of hydrophobic substrates to the catalytic active site and holding P450 in an orientation that allows optimal contact with its redox partners for efficient electron transfer (
      • Williams P.A.
      • Cosme J.
      • Sridhar V.
      • Johnson E.F.
      • McRee D.E.
      Mammalian microsomal cytochrome P450 monooxygenase.
      ,
      • Baylon J.L.
      • Lenov I.L.
      • Sligar S.G.
      • Tajkhorshid E.
      Characterizing the membrane-bound state of cytochrome P450 3A4: structure, depth of insertion, and orientation.
      ). Furthermore, it is reported that substrate turnover could be stimulated if phospholipids are present (
      • Strobel H.W.
      • Lu A.Y.H.
      • Heidema J.
      • Coon M.J.
      Phosphatidylcholine requirement in the enzymatic reduction of hemoprotein P-450 and in fatty acid, hydrocarbon, and drug hydroxylation.
      ,
      • van der Hoeven T.A.
      • Coon M.J.
      Preparation and properties of partially purified cytochrome P-450 and reduced nicotinamide adenine dinucleotide phosphate-cytochrome P-450 reductase from rabbit liver microsomes.
      ). Extensive studies on the interaction and electron transfer between P450 and CPR in the presence and absence of phospholipids have been carried out and revealed stronger interactions between the two proteins and faster electron transfer from CPR to P450 in the presence of lipids or membrane mimetics (
      • Müller-Enoch D.
      • Churchill P.
      • Fleischer S.
      • Guengerich F.P.
      Interaction of liver microsomal cytochrome P-450 and NADPH-cytochrome P-450 reductase in the presence and absence of lipid.
      • Miwa G.T.
      • Lu A.Y.H.
      Studies on the stimulation of cytochrome P-450-dependent monooxygenase activity by dilauroylphosphatidylcholine.
      ,
      • Coon M.J.
      • Haugen D.A.
      • Guengerich F.P.
      • Vermilion J.L.
      • Dean W.L.
      • Imaoka S.
      • Imai Y.
      • Shimada T.
      • Funae Y.
      Role of phospholipids in reconstituted cytochrome P450 3A form and mechanism of their activation of catalytic activity.
      ). However, studies demonstrating the effect of phospholipid membrane on P450-cytb5 interactions are still lacking in the literature.
      Although cytb5 is only capable of donating the second electron due to its high redox potential as compared with ferric P450, it plays a key role in the P450 enzyme system in the catalysis of a variety of compounds and significantly regulates the functional activities of P450s (
      • Kominami S.
      • Ogawa N.
      • Morimune R.
      • D-Ying H.
      • Takemori S.
      The role of cytochrome b5 in adrenal microsomal steroidogenesis.
      ,
      • Finn R.D.
      • McLaughlin L.A.
      • Ronseaux S.
      • Rosewell I.
      • Houston J.B.
      • Henderson C.J.
      • Wolf C.R.
      Defining the in vivo role for cytochrome b5 in cytochrome P450 function through the conditional hepatic deletion of microsomal cytochrome b5.
      ). It is reported that cytb5 could stimulate, inhibit, or have no effect on P450 activities, depending on the P450 isozyme studied, the substrate involved, or the particular experimental conditions employed (
      • Morgan E.T.
      • Coon M.J.
      Effects of cytochrome b5 on cytochrome P-450-catalyzed reactions: studies with manganese-substituted cytochrome b5.
      • Canova-Davis E.
      • Chiang J.Y.L.
      • Waskell L.
      Obligatory role of cytochrome b5 in the microsomal metabolism of methoxyflurane.
      ,
      • Shimada T.
      • Mernaugh R.L.
      • Guengerich F.P.
      Interactions of mammalian cytochrome P450, NADPH-cytochrome P450 reductase, and cytochrome b5 enzymes.
      • Im S.-C.
      • Waskell L.
      The interaction of microsomal cytochrome P450 2B4 with its redox partners, cytochrome P450 reductase and cytochrome b5.
      ). The mechanism underlying the differential effects of cytb5 on P450 activities is not fully understood. A generally well accepted explanation is that cytb5 and CPR possess an overlapping but non-identical binding surface on P450, resulting in competitive binding between the two proteins (
      • Estrada D.F.
      • Laurence J.S.
      • Scott E.E.
      Substrate-modulated cytochrome P450 17A1 and cytochrome b5 interactions revealed by NMR.
      ,
      • Bridges A.
      • Gruenke L.
      • Chang Y.T.
      • Vakser I.A.
      • Loew G.
      • Waskell L.
      Identification of the binding site on cytochrome P450 2B4 for cytochrome b5 and cytochrome P450 reductase.
      ). When P450 predominantly binds cytb5 due to a high cytb5 concentration, the first electron to be delivered from CPR is inhibited (
      • Dürr U.H.N.
      • Waskell L.
      • Ramamoorthy A.
      The cytochromes P450 and b5 and their reductases: promising targets for structural studies by advanced solid-state NMR spectroscopy.
      ,
      • Bridges A.
      • Gruenke L.
      • Chang Y.T.
      • Vakser I.A.
      • Loew G.
      • Waskell L.
      Identification of the binding site on cytochrome P450 2B4 for cytochrome b5 and cytochrome P450 reductase.
      ). To obtain an insight into the influence of cytb5 on P450 activities, an in-depth understanding of P450-cytb5 interaction is necessary. A recent study on the interaction between the truncated cytochrome P450 17A1 and the soluble domain of human cytb5 in a lipid-free environment revealed a binding interface located on the upper cleft of cytb5 (
      • Estrada D.F.
      • Laurence J.S.
      • Scott E.E.
      Substrate-modulated cytochrome P450 17A1 and cytochrome b5 interactions revealed by NMR.
      ). Because both proteins are naturally membrane-bound, our study aims to characterize the interactions of P450 and cytb5 in a native-like membrane environment in order to obtain a more physiologically relevant view. Although the microsomes resemble the ER membrane most, it is a very complex system containing a variety of different types of lipids, cholesterol, carbohydrates, proteins, etc. (
      • DePierre J.W.
      • Ernster L.
      Enzyme topology of intracellular membranes.
      ,
      • Depierre J.W.
      • Dallner G.
      Structural aspects of the membrane of the endoplasmic reticulum.
      ) For mechanistic studies on the P450 system, a model membrane is needed that can both mimic the native membrane environment and be easily characterized and controlled. Among the most suitable membrane mimetics for NMR studies, detergent micelles and phospholipid/detergent isotropic bicelles have been most frequently and successfully applied to the investigation of the structure and function of a number of different membrane proteins during the past few decades, as reviewed in Refs.
      • Dürr U.H.N.
      • Gildenberg M.
      • Ramamoorthy A.
      The magic of bicelles lights up membrane protein structure.
      and
      • Arora A.
      • Tamm L.K.
      Biophysical approaches to membrane protein structure determination.
      .
      In this study, we report an investigation of the interaction between full-length rabbit cytochrome P450 2B4 (CYP2B4) and full-length rabbit cytb5 in different membrane mimetic environments, including lipid-free environment and isotropic bicelles and micelles, utilizing NMR techniques. Our study provides the first structural evidence on the importance of the phospholipid bilayer in governing the interaction between these two proteins. The mechanism by which the membrane affects CYP2B4-cytb5 interaction is also explored. By comparing the effects of different membrane environments in assisting complex formation, we propose that phospholipid bilayers enhance both the affinity and specificity in the interaction between CYP2B4 and cytb5.

      Acknowledgments

      We thank Dr. Patrick Walsh for critical reading of the manuscript.

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