Membrane-Protein Interactions Contribute to Efficient 27-Hydroxylation of Cholesterol by Mitochondrial Cytochrome P450 27A1

Murtazina, Dilyara A.; Puchkaev, A. V.; Schein, Catherine H.; Oezguen, Numan; Braun, Werner; Nanavati, Amit A.; Pikuleva, Irina A.

doi:10.1074/jbc.m204909200

Cited by 45 publications

(41 citation statements)

References 24 publications

(28 reference statements)

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“…1D, 2C, and 3). It is consistent with mutagenesis experiments showing this region is important for membrane binding (33,41,42), fluorescence quenching analysis (5), the predicted pose of aromatase (36), and computational simulations for human cytochrome P450s performed in the presence of diolyeolphosphatidylcholine (DOPC) (37) or 1-palmitoyl-2-oleoyl-sn-glycerol-2-phosphocholine (POPC) (27, 32-34, 37, 43). Several aspects of the structure support the proposed orientation.…”

Section: Resultssupporting

confidence: 61%

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.

248

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: Resultssupporting

confidence: 61%

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.

248

283

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“…This phenomenon may be an artifact of our heterologous expression system, as mutagenesis of analogous hydrophilic domains has not been precluded for other CYPs, including CYP51 [35] and the more closely related cholesterol metabolizing CYPs, CYP27A1 [36,37] and CYP7A1 [42]. Our modeling and biochemical studies suggests that proper organization of the B'-helix of CYP24A1, through contacts with the heme, F, G and I-helices, and the F-G and B'-C loops is required to maintain structural integrity of the reactive heme center.…”

Section: Discussionmentioning

confidence: 99%

Hybrid homology modeling and mutational analysis of cytochrome P450C24A1 (CYP24A1) of the Vitamin D pathway: Insights into substrate specificity and membrane bound structure–function

Annalora

Bobrovnikov-Marjon

Serda

et al. 2007

Archives of Biochemistry and Biophysics

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Cytochrome P450C24A1 (CYP24A1), a peripheral inner mitochondrial membrane hemoprotein and candidate oncogene, regulates the side-chain metabolism and biological function of vitamin D and many of its related analog drugs. Rational mutational analysis of rat CYP24A1 based on hybrid (2C5/ BM-3) homology modeling and affinity labeling studies clarified the role of key domains (Nterminus, A', A, and F-helices, β3a strand, & β5 hairpin) in substrate binding and catalysis. The scope of our study was limited by an inability to purify stable mutant enzyme targeting soluble domains (B', G, and I-helices) and suggested greater conformational flexibility among CYP24A1's membraneassociated domains. The most notable mutants developed by modeling were V391T and I500A, which displayed defective binding function and profound metabolic defects for 25-hydroxylated vitamin D 3 substrates similar to a non-functional F-helix mutant (F249T) that we previously reported. Val-391 (β3a strand) and Ile-500 (β5 hairpin) are modeled to interact with Phe-249 (F-helix) in a hydrophobic cluster that directs substrate binding events through interactions with the vitamin D cis-triene moiety. Prior affinity labeling studies identified an amino-terminal residue (Ser-57) as a putative active-site residue that interacts with the 3β-OH group of the vitamin D A-ring. Studies with 3-epi and 3-deoxy-1,25(OH) 2 D 3 analogs confirmed interactions between the 3β-OH group and Ser-57 effect substrate recognition and trafficking while establishing that the trans conformation of A-ring hydroxyl groups (1α & 3β) is obligate for high-affinity binding to rat CYP24A1. Our work suggests that CYP24A1's amphipathic nature allows for monotopic membrane insertion, whereby a pw2d-like substrate access channel is formed to shuttle secosteroid substrate from the membrane to the active-site. We hypothesize that CYP24A1 has evolved a unique amino-terminal membrane § Deceased

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“…The fourth P450 is CYP11A1, which catalyzes the first step in overall steroid hormone biosynthesis in steroidogenic tissues. CYP7A1, CYP27A1, CYP46A1, and CYP11A1 bind cholesterol with nanomolar to low micromolar constants (the K d value depends on whether detergent is present and at what concentration in the assay buffer) (29,30,33) but metabolize it to different products: 7 ␣ -hydroxycholesterol (CYP7A1), 27-hydroxycholesterol (CYP27A1), 24 S -hydroxycholesterol (CYP46A1), and pregnenolone (CYP11A1). Cholesterol-metabolizing P450s belong to the P450 superfamily of enzymes that contain an iron protoporphyrin IX as a prosthetic group (34).…”

mentioning

confidence: 99%

“…Several different approaches were used to evaluate the ability of these proteins to bind cholesterol. They were based on: 1 ) use of a fluorescent cholesterol derivative that changes its fluorescence when bound to a protein (25); 2 ) immunostaining of a protein bound to either a TLC or a microtiter plate coated with cholesterol (26,27); 3 ) detection of a spectral change in a protein induced by cholesterol binding (28)(29)(30); and 4 ) radioactivity measurements after a protein was incubated with a mixture of radiolabeled and unlabeled cholesterol and separated from unbound cholesterol either via affinity or other types of classic column chromatography (18,31) or via centrifugation of the assay mixture through microcolumns (14,32).…”

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confidence: 99%

A simple and rapid method to measure cholesterol binding to P450s and other proteins

Mast

Pikuleva

2005

Journal of Lipid Research

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Cholesterol plays an important role in cellular function and membrane compartmentalization and is involved in the interaction with more than a dozen of different proteins. Using three cholesterol-metabolizing cytochrome P450s (P450s 7A1, 46A1, and 11A1), we have developed a rapid and simple assay for measurements of nanomolar to micromolar cholesterol affinities. In this assay, the P450 is incubated with a fixed amount of radiolabeled cholesterol and varying concentrations of cold cholesterol followed by separation of free and protein-bound cholesterol via filtration through a membrane. Free cholesterol is found in the flowthrough fraction, whereas P450 binds to the membrane. The radioactivity of the membranes is then measured, and a saturation curve is generated after correction for nonspecific binding of cholesterol to the filter. The validity of the filter assay was confirmed by spectral assay, a traditional method to evaluate the interaction of the P450 enzymes with their substrates. Two types of membranes, one binding positively charged proteins and another binding negatively charged proteins, were identified. These membranes were also found to hold proteins through hydrophobic interactions. Thus, the cholesterol binding properties of a wide variety of proteins could be characterized using this filter assay.

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Membrane-Protein Interactions Contribute to Efficient 27-Hydroxylation of Cholesterol by Mitochondrial Cytochrome P450 27A1

Cited by 45 publications

References 24 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

Hybrid homology modeling and mutational analysis of cytochrome P450C24A1 (CYP24A1) of the Vitamin D pathway: Insights into substrate specificity and membrane bound structure–function

A simple and rapid method to measure cholesterol binding to P450s and other proteins

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