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.

Fig. 1.

Overall fold of ScErg11p and predicted orientation in the lipid bilayer. (A) Helices are colored from the N terminus to the C terminus with a gradient from blue to red. The heme is shown as yellow sticks; lanosterol in the active-site cavity is shown in orange. (B) The structure of the amphipathic helix (MH1) and the transmembrane helix (TMH1). Hydrophobic side chains are colored blue, and polar residues colored magenta, with oxygen atoms colored red, and nitrogen atoms colored blue. Difference electron density (Fo–Fc) contoured to 2σ is depicted as a green mesh. (C) Interaction of the transmembrane domain with the catalytic domain. Ordered water molecules are depicted as red spheres. Hydrogen bonding interactions are shown as dashed lines; distances are in Å. (D) Distribution of hydrophobic and charged residues in the F/G loop.

Fig. 2.

Crystal packing of ScErg11p-6xHis. (A) ScErg11p-6xHis crystals show alternating layers of transmembrane (TM) segments and the soluble segments (S). (B) Crystal packing of two symmetry-related copies of ScErg11p-6xHis with the amphipathic helix S6–H21. The amphipathic helix is colored green, and the symmetrically related copies are colored salmon or blue. (C) Overlay of lanosterol-bound (gray) and ITC-bound (salmon) ScErg11p-6xHis.

Fig. 3.

Sequence and predicted membrane interactions of ScErg11p. (A) Regions highlighted in yellow are predicted by OPM to lie inside the lipid bilayer. Red squares indicate acidic residues that were accessible to the water-soluble glycinamide probe when ScErg11p-6xHis was incorporated into artificial liposomes. (B) Electrostatic surface map of ScErg11p with cutaway showing the primary and secondary vestibules. Surface electrostatic potential is displayed with the molecular graphics software Chimera (59) with blue as positive and red as negative.

Fig. 4.

Lanosterol and ITC binding in ScErg11p. (A) Lanosterol is depicted with carbon atoms colored cyan and heme with carbon atoms colored yellow. Selected oxygen atoms are colored red, nitrogen blue, sulfur gold, and iron brown. Electron density is depicted as a simulated annealing 2Fo–Fc omit map contoured to 0.7σ with the ligand, heme, and oxygen omitted from the electron density calculation. A bond links the heme–oxo complex. (B) ITC in a coordination complex with active-site heme. The color scheme is as in A, with ITC carbons colored magenta. Electron density is depicted as an Fo–Fc map contoured to 2.5σ with the ligand density omitted from the calculation. (C) Conserved amino acids in fungi that are commonly mutated in antifungal resistance are depicted with carbon atoms colored orange. Amino acids conserved in S. cerevisiae and in the main fungal pathogens of humans but not in human CYP51, and not mutated, are depicted with carbon atoms colored green. Amino acids that are similar only in the pathogenic fungi are shown in cyan. Other atoms are colored as in A.

Fig. 5.

Multiple ligands bind to ScErg11p. (A) Electron density of lanosterol-like molecule. Fo–Fc density contoured to 2.0σ is shown in green with a theoretical model of 4,4-dimethyl-cholesta-8,14,24-trienol placed. The 4,4-dimethyl-cholesta-8,14,24-trienol is depicted with carbon atoms colored cyan. (B) Hypothetical model of ligand binding with surface representation of the primary and secondary vestibules. Lanosterol bound in the active site is depicted with carbon atoms colored cyan. The second lanosterol-like molecule is depicted with carbon atoms colored purple. Oxygen atoms are colored red, nitrogen blue, and iron brown. The outside surface of the vestibules and active site are depicted as a light mesh surface colored dark gray, and their visible inside surface is colored light gray.