Structural Insights into Binding of the Antifungal Drug Fluconazole to Saccharomyces cerevisiae Lanosterol 14α-Demethylase

Abstract

Infections by fungal pathogens such as Candida albicans and Aspergillus fumigatus and their resistance to triazole drugs are major concerns. Fungal lanosterol 14α-demethylase belongs to the CYP51 class in the cytochrome P450 superfamily of enzymes. This monospanning bitopic membrane protein is involved in ergosterol biosynthesis and is the primary target of azole antifungal drugs, including fluconazole. The lack of high-resolution structural information for this drug target from fungal pathogens has been a limiting factor for the design of modified triazole drugs that will overcome resistance. Here we report the X-ray structure of full-length Saccharomyces cerevisiae lanosterol 14α-demethylase in complex with fluconazole at a resolution of 2.05 Å. This structure shows the key interactions involved in fluconazole binding and provides insight into resistance mechanisms by revealing a water-mediated hydrogen bonding network between the drug and tyrosine 140, a residue frequently found mutated to histidine or phenylalanine in resistant clinical isolates.

FIG 1

The chemical structures of the substrate lanosterol, the short-tailed triazole antifungal agents FLC and VCZ, and the long-tailed azole antifungal ITC. An asterisk (*) indicates the 14α-methyl of the lanosterol.

FIG 2

The structure of ScErg11p6×His. The protein is colored from the N terminus to the C terminus with a gradient from blue to red. The heme (purple carbons) and FLC (green carbons) are shown in sticks. For clarity, only some of the α-helices and β-sheets are labeled. FSL, fungus-specific loop.

FIG 3

Spectral characterization of ScErg11p6×His. Data represent the absolute spectra of the ferric protein (solid trace), the ferrous protein reduced with sodium dithionite (dashed trace), and the reduced protein with bound carbon monoxide (dotted trace). Spectra were recorded using 1.5 μM ScErg11p as described in Materials and Methods.

FIG 4

Binding of FLC and VCZ to ScErg11p6×His. (a) Difference spectra demonstrating type II binding of FLC obtained by incremental additions of the drug to 1 μM ScErg11p×His. (b) Saturation curves for FLC (filled circles) and VCZ (hollow circles) fitted to the Hill equation, with ΔA plotted against azole concentration.

FIG 5

FLC binding in the active site of ScErg11p. (a) OMIT map for FLC (F_o-F_c map [green mesh] contoured at 3σ; 2F_o-F_c map [blue mesh] contoured at 1σ). The F_o-F_c map was calculated using F_calc refined from coordinates with no ligand at the active site. The 2F_o-F_c map was calculated following the final refinement. The main chain is indicated in gray, and fluconazole is indicated as sticks, with C atoms in cyan, N atoms blue, O atoms in red, and F atoms in pale blue. The heme is shown as sticks, with C atoms colored magenta. (b) Side chains of amino acid residues within 4 Å of FLC are indicated in gray. The main chain atoms are shown for G310, G314, and G315. (c) Water-mediated hydrogen bonding (yellow dashed lines) between HOH743, FLC, heme, and Y140, as well as between HOH790, FLC, and S382.