Membrane Topology of Cytochrome P450 2B4 in Langmuir–Blodgett Monolayers

Shank-Retzlaff, Mary; Raner, Gregory M.; Coon, Minor J.; Sligar, Stephen G.

doi:10.1006/abbi.1998.0889

Cited by 24 publications

(19 citation statements)

References 33 publications

(29 reference statements)

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“…About 30% of the catalytic domain of CYP11A1 was inaccessible to chemical modification by fluorescent probes (33). The physical displacement of phospholipids in Langmuir-Blodgett monolayers indicated a considerably greater area of protein insertion than could be accounted for by a single N-terminal transmembrane helix (34). This finding was supported by atomic force microscopy, which showed the catalytic domain protrudes only 3.5 ± 1 nm above the membrane (35).…”

Section: Significancementioning

confidence: 87%

Architecture of a single membrane spanning cytochrome P450 suggests constraints that orient the catalytic domain relative to a bilayer

Monk

Tomasiak

Keniya

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

244

283

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Bitopic integral membrane proteins with a single transmembrane helix play diverse roles in catalysis, cell signaling, and morphogenesis. Complete monospanning protein structures are needed to show how interaction between the transmembrane helix and catalytic domain might influence association with the membrane and function. We report crystal structures of full-length Saccharomyces cerevisiae lanosterol 14α-demethylase, a membrane monospanning cytochrome P450 of the CYP51 family that catalyzes the first postcyclization step in ergosterol biosynthesis and is inhibited by triazole drugs. The structures reveal a well-ordered N-terminal amphipathic helix preceding a putative transmembrane helix that would constrain the catalytic domain orientation to lie partly in the lipid bilayer. The structures locate the substrate lanosterol, identify putative substrate and product channels, and reveal constrained interactions with triazole antifungal drugs that are important for drug design and understanding drug resistance.M embrane proteins that span the lipid bilayer once constitute around 50% of all integral membrane proteins (1). Although monospanning membrane proteins carry out numerous key biological functions, including environmental sensing, organellespecific catalysis, and the regulation of cell morphology, only individual domains or subdomains are currently represented in the Protein Data Bank, and structural information about interactions between their transmembrane domains and extramembranous components is lacking. Cytochrome P450 proteins are prominent enzymes with orthologs found in all kingdoms of life. In eukaryotes, microsomal members of this major family of mixed-function mono-oxygenases contain a single transmembrane helix and can be grouped in two broad functional categories: biodefense, such as the first phase of xenobiotic detoxification, and core metabolism including reactions in sterol biosynthesis and fatty acid oxidation (2).The lanosterol 14α-demethylases or CYP51 enzymes, probably the most genetically ancient of the cytochrome P450 families, play a central role in cholesterol or ergosterol biosynthesis (3). CYP51s carry out three consecutive mono-oxygenase reaction cycles to remove the 14α-methyl group from lanosterol to yield 4,4-dimethyl-cholesta-8,14,24-trienol, a key precursor in cholesterol and ergosterol biosynthesis, releasing water and formic acid (3). Because of the key roles that CYP51s play in yeast, filamentous fungi, and some parasitic protozoa, these enzymes are therapeutic targets for antimicrobial agents, including fluconazole (FLC), voriconazole (VCZ), and itraconazole (ITC) (4). Fungal infections play an increasingly significant role in disease, impacting agriculture ecosystems and human health, especially in immunocompromised individuals (5-7) for whom antifungal resistance continually poses a threat (8). In humans CYP51 is being tested as a target for cholesterol-lowering drugs (9) and in antiangiogenic cancer therapies (10). A limited set of cytochrome P450 isoforms (1A2, 2C8, 2C...

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Section: Significancementioning

confidence: 87%

Architecture of a single membrane spanning cytochrome P450 suggests constraints that orient the catalytic domain relative to a bilayer

Monk

Tomasiak

Keniya

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

244

283

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“…Both tr-CYP2B4 and a num- ber of other P450 isoforms lacking the NH 2 -terminal membrane anchor region expressed in bacteria (17)(18)(19)(20), yeast (21) and mammalian cells (22) have been shown to retain much of their membrane localizations, suggesting that the TM domain is not the sole determinant for membrane binding and that direct interaction exists between the tr-P450s and the membrane. Both an atomic force microscopy study (64) and a Langmuir-Blodgett monolayer study (69) have suggested that a segment of the tr-P450 is buried in the membrane; the observed slow rotation rate for membrane-embedded cytochrome P450s could be well explained by a secondary binding interaction between the tr-P450 and the membrane (62,63). A recent simulation study on CYP3A4 also supports partial insertion of the tr-P450 into a membrane mimetic (25).…”

Section: Effect Of Dlpc/dhpc Isotropic Bicelles On Cyp2b4-cytbmentioning

confidence: 99%

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

Zhang

Huang

et al. 2015

Journal of Biological Chemistry

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Background:The functional activities of membrane-bound cytochrome P450s (P450s) are affected by lipids. Results: Stronger interactions between P450 2B4 (CYP2B4) and cytochrome b 5 (cytb 5 ) are observed in bicelles as compared with lipid-free solution or micelles. Conclusion: Lipid bilayers enhance the interaction between CYP2B4 and cytb 5 . Significance: The findings provide insights into the important role of membrane in P450-cytb 5 complex formation.

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“…Coon et al (9) first reported the isolation of 2B4, originally named LM2, from phenobarbital-induced rabbit liver microsomes in 1973. Since that time, 2B4 has served as an experimental model for biochemical and biophysical studies of mammalian P450 interactions with lipid (10)(11)(12) and the redox partners NADPH-P450 reductase and cytochrome b 5 (13), as well as mechanistic studies (14)(15)(16). Many of these studies, and the extraordinary wealth of site-directed mutagenesis data (17), suggest that a closed conformation would be most compatible with metabolism once substrate is bound in the active site.…”

mentioning

confidence: 99%

An open conformation of mammalian cytochrome P450 2B4 at 1.6-Å resolution

Scott

Wester

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

339

358

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The xenobiotic metabolizing cytochromes P450 (P450s) are among the most versatile biological catalysts known, but knowledge of the structural basis for their broad substrate specificity has been limited. P450 2B4 has been frequently used as an experimental model for biochemical and biophysical studies of these membrane proteins. A 1.6-Å crystal structure of P450 2B4 reveals a large open cleft that extends from the protein surface directly to the heme iron between the ␣-helical and ␤-sheet domains without perturbing the overall P450 fold. This cleft is primarily formed by helices B to C and F to G. The conformation of these regions is dramatically different from that of the other structurally defined mammalian P450, 2C5/3LVdH, in which the F to G and B to C regions encapsulate one side of the active site to produce a closed form of the enzyme. The open conformation of 2B4 is trapped by reversible formation of a homodimer in which the residues between helices F and G of one molecule partially fill the open cleft of a symmetryrelated molecule, and an intermolecular coordinate bond occurs between H226 and the heme iron. This dimer is observed both in solution and in the crystal. Differences between the structures of 2C5 and 2B4 suggest that defined regions of xenobiotic metabolizing P450s may adopt a substantial range of energetically accessible conformations without perturbing the overall fold. This conformational flexibility is likely to facilitate substrate access, metabolic versatility, and product egress. T he cytochromes P450 (P450s) are a superfamily of hemecontaining monooxygenases. They are responsible for the metabolism of an unusually wide range of endogenous and exogenous substrates, including synthesis of steroid hormones, bile acids, and cholesterol, and the degradation of steroids, fatty acids, drugs, toxins, and procarcinogens (1). P450s from families 1, 2, and 3 have evolved to convert lipophilic xenobiotics to more polar metabolites readily conjugated by phase II enzymes, and thus targeted for elimination. The stereo-and regiospecificity of metabolite formation by individual xenobiotic metabolizing P450s suggest very specific substrate-enzyme interactions, whereas the range of substrates metabolized suggests an induced fit type of substrate recognition. Understanding the basis for this specific, yet versatile, metabolism by mammalian xenobiotic metabolizing P450s has been limited by the dearth of structural information for these membrane proteins.In 2000, the first mammalian P450 structure was published (2, 3), that of P450 2C5, which was engineered to delete the single N-terminal transmembrane domain and to mutate a peripheral membrane-binding site. Recent structures of 2C5 bound with the substrates, diclofenac (4) and 4-methyl-N-methyl-N-(2-phenyl-2H-pyrazol-3-yl)benzenesulfonamide (DMZ) (5), indicate that flexible regions of the protein adapt for substrate binding, and that ligands may bind in multiple orientations. The 2C5 structures generally reveal the enzyme closed around these substrates wi...

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Membrane Topology of Cytochrome P450 2B4 in Langmuir–Blodgett Monolayers

Cited by 24 publications

References 33 publications

Architecture of a single membrane spanning cytochrome P450 suggests constraints that orient the catalytic domain relative to a bilayer

Architecture of a single membrane spanning cytochrome P450 suggests constraints that orient the catalytic domain relative to a bilayer

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

An open conformation of mammalian cytochrome P450 2B4 at 1.6-Å resolution

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