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

Abstract

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...

The Crystal Structure of Cytochrome P450 4B1 (CYP4B1) Monooxygenase Complexed with Octane Discloses Several Structural Adaptations for ω-Hydroxylation

“…The trajectory of the AЉ helix differs from that observed for Saccharomyces cerevisiae Cyp51A1 (Fig. 2C), which was crystallized with its complete N-terminal region (38). In the CYP51A1 structure, the shorter polar linker region makes a more acute turn between the catalytic domain and the hydrophobic TMH AЉ, which passes on the side of the first turn in ␤-sheet 1 that is opposite from the path of the AЉ helix seen in CYP4B1 (Fig.…”

Interactions among Cytochromes P450 in Microsomal Membranes

“…This conclusion is in agreement with the results of Baylon et al (55), who concluded that the membrane binding of P450 is largely independent of the presence of the N-terminal hydrophobic anchor. According to the current concepts, the interactions of the microsomal P450 enzymes with the membrane are in large part determined by the regions between the N-terminal anchor and ␣-helix A (54,56,57) as well as by the hydrophobic surface in the region of ␣-helices FЈ and GЈ (55,58,59). Furthermore, according to the x-ray structure of the full-length CYP51, the constrained orientation of the P450 catalytic domain relative to the membrane is dictated by a network of interactions of polar residues in the C-terminal part of the transmembrane helix and the following proline-rich region (54), which are completely retained in our constructs.…”

Structural characterization of human cholesterol 7α-hydroxylase

“…The meander is located close to the Cys-binding loop, which can modulate electronic properties of the heme and ultimately the catalytic function of CYP7A1 ( 44 ). An alternative positioning of the meander is observed in full-length yeast CYP51 in complex with lanosterol ( 45 ) and suggests the following: a ) a signifi cant fl exibility of this region that enables its relocation for capping the Cys-binding loop and contacts with the C helix, thereby dramatically changing the electron transfer interface; and b ) that it may interact with other protein effectors, which stimulate/regulate substrate binding. Modulation of the interaction interface by the longer meander might also be a feature of CYP7/8 proteins evolutionary closest to CYP51 and might be further adapted for nonmonooxygenase chemistry (e.g., selfsuffi cient CYP8A1).…”