Membrane Position of Ibuprofen Agrees with Suggested Access Path Entrance to Cytochrome P450 2C9 Active Site

Berka, Karel; Hendrychová, Tereza; Anzenbacher, Pavel; Otyepka, Michal

doi:10.1021/jp204488j

Cited by 131 publications

(173 citation statements)

References 67 publications

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“…Further positioning in the active site would ensure that the position of cholesterol hydroxylation approaches the heme iron. Consistent with a generally accepted model for cholesterol substrate delivery directly from the membrane, this relatively straightforward binding model is supported by mutagenesis, chemical modifi cation studies ( 28,30,38 ), and membrane penetration depth calculations (39)(40)(41). Interestingly, all available mammalian P450s structures of enzymes that hydroxylate sterol substrates (CYP11A1: PDB code 3N9Y, CYP46A1: PDB code 2Q9F, and CYP7A1: PDB code 3SN5), independent of the cholesterol hydroxylation position on the aliphatic chain or the ring system, show a conserved cholesterol orientation (side chain near the heme).…”

Section: Substrate Recognitionsupporting

confidence: 64%

Structural characterization of human cholesterol 7α-hydroxylase

Tempel

Grabovec

Mackenzie

et al. 2014

Journal of Lipid Research

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Section: Substrate Recognitionsupporting

confidence: 64%

Structural characterization of human cholesterol 7α-hydroxylase

Tempel

Grabovec

Mackenzie

et al. 2014

Journal of Lipid Research

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“…A crystal structure of full-length aromatase at 2.9-Å resolution has been modeled, but weak electron density meant that the structures of 44 N-terminal and seven C-terminal residues could not be determined (36). Models that place the mouth of the substrate channel in direct contact with the hydrophobic environment of the lipid bilayer required the catalytic domain helices A′ and A to be imbedded and partially imbedded, respectively, in the lipid bilayer (28,37).…”

Section: Significancementioning

confidence: 99%

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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“…5 in Berka et al, 2011). Two independent models of membrane anchored CYP2C9 based on molecular dynamics simulations (Berka et al, 2011;Cojocaru et al, 2011) published in 2011 shared many similarities indicating that molecular dynamics might provide convergent and reliable membrane Cyp3a null (open triangle) mice (female; n = 4) were fed on a powdered diet (RM1) containing 250 mg/kg pregnenolone-16a-carbonitrile (PCN) for 3 days (equivalent to 50 mg/kg daily). On day 4, mice were dosed p.o.…”

Section: Positioning Of Microsomal P450s and Drugs In Lipid Bilayersmentioning

confidence: 99%

The Role of Protein-Protein and Protein-Membrane Interactions on P450 Function

Scott

Wolf

Otyepka

et al. 2016

Drug Metabolism and Disposition

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This symposium summary, sponsored by the ASPET, was held at Experimental Biology 2015 on March 29, 2015, in Boston, Massachusetts. The symposium focused on: 1) the interactions of cytochrome P450s (P450s) with their redox partners; and 2) the role of the lipid membrane in their orientation and stabilization. Two presentations discussed the interactions of P450s with NADPH-P450 reductase (CPR) and cytochrome b 5 . First, solution nuclear magnetic resonance was used to compare the protein interactions that facilitated either the hydroxylase or lyase activities of CYP17A1. The lyase interaction was stimulated by the presence of b 5 and 17a-hydroxypregnenolone, whereas the hydroxylase reaction was predominant in the absence of b 5 . The role of b 5 was also shown in vivo by selective hepatic knockout of b 5 from mice expressing CYP3A4 and CYP2D6; the lack of b 5 caused a decrease in the clearance of several substrates. The role of the membrane on P450 orientation was examined using computational methods, showing that the proximal region of the P450 molecule faced the aqueous phase. The distal region, containing the substrate-access channel, was associated with the membrane. The interaction of NADPH-P450 reductase (CPR) with the membrane was also described, showing the ability of CPR to "helicopter" above the membrane. Finally, the endoplasmic reticulum (ER) was shown to be heterogeneous, having ordered membrane regions containing cholesterol and more disordered regions. Interestingly, two closely related P450s, CYP1A1 and CYP1A2, resided in different regions of the ER. The structural characteristics of their localization were examined. These studies emphasize the importance of P450 protein organization to their function.

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Membrane Position of Ibuprofen Agrees with Suggested Access Path Entrance to Cytochrome P450 2C9 Active Site

Cited by 131 publications

References 67 publications

Structural characterization of human cholesterol 7α-hydroxylase

Structural characterization of human cholesterol 7α-hydroxylase

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

The Role of Protein-Protein and Protein-Membrane Interactions on P450 Function

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