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

Abstract

cInfections by fungal pathogens such as Candida albicans and Aspergillus fumigatus and their resistance to triazole drugs are major concerns. Fungal lanosterol 14␣-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 14␣-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.

Proposal for a unified nomenclature for target‐site mutations associated with resistance to fungicides

“…Amino acid sequences were downloaded from NCBI GenBank and annotated with reported amino acid substitutions8, 12, 13, 14, 15 using Geneious 6.1.8 software (Biomatters). Alignments of sequences were generated using the ClustalW16 algorithm with Blosum scoring matrix, gap opening penalty 10, gap extension penalty 0.5 and free end gaps.…”

Structural and Functional Elucidation of Yeast Lanosterol 14α-Demethylase in Complex with Agrochemical Antifungals

“…The hydroxyl group of both enantiomers point in the same direction toward Y126 and Y140. The position of these hydroxyl groups differs by 1.6 Å due to the large 4-chlorophenethyl group of S -TBZ (bounded by G310 in helix I and I139 in the loop joining helices B and C; Fig 3C) projecting in the active site to the position occupied by the disubstituted phenyl ring of FLC (PDB ID:4WMZ) [21]. The smaller tertiary butyl group of S -TBZ is positioned towards the substrate entry channel.…”

“…The central quaternary carbons of the enantiomers are approximately 1 Å apart, with R -TBZ projecting deeper into the active site cavity which easily accommodates the smaller tert -butyl substituent (Fig 3C). The key water 743 identified in the wild type FLC structure [21, 22] is not seen in either TBZ structure. This may be due to the lower resolution of this structure but could easily be accommodated.…”

“…The 2-chlorophenyl ring of the R enantiomer occupies the deep cavity similar to FLC with the chlorocyclopropyl group occupying the larger cavity towards the entry channel. Conversely, the chlorocyclopropyl group of the more active S enantiomer (see below) is situated in the deep cavity distal to the entry channel with the 2-chlorophenyl ring occupying the cavity towards the entry channel and binds in the same plane as the non-coordinating triazole of FLC in the wild type structure [21]. The 2-chloro substituent of the phenyl ring is in close proximity to the phenyl ring of Phe236, causing it to move away from the inhibitor compared with the other enantiomer.…”

“…The absolute absorbance spectra for Ni-NTA affinity and SEC purified ScErg11p6×His showed a Soret peak at 417 nm [21]. Type II azole binding is characterised by a shift of the Soret peak from 417 nm to 421–424 nm in ScErg11p6×His and other cytochrome P450s.…”