The monospanning membrane protein lanosterol 14α-demethylase (Erg11p) catalyses the rate limiting step of ergosterol biosynthesis in fungi and is the target of triazole drugs. Inhibition of Erg11p depletes cell membranes of ergosterol, inhibiting fungal growth. Resistance to triazoles can arise due to mutations in Erg11p that reduce the binding affinity of the drug.
Objective: This project aimed to understand how mutations in Erg11p affect the binding of triazoles.
Methods: Mutations Y140F and G73E were introduced into
S. cerevisiae Erg11p6×His (ScErg11p) overexpressed from the
PDR5 locus of yeast strain AD2Δ. The endogenous
ERG11 was deleted. ScErg11p was solubilised with N-decyl-β-D-maltoside and purified by Ni-NTA affinity chromatography and size exclusion chromatography using a Superdex 200™ column.
Results: X-ray structures of ScErg11p Y140F and G73E mutants in complex with itraconazole (ITC) and wild type ScErg11p and the Y140F mutant in complex with fluconazole (FLC) were determined. The
Candida albicans Erg11p Y132F mutation in clinical isolates and the equivalent Y140F mutation in ScErg11p conferred resistance to FLC. The
Aspergillus fumigatus Erg11p G54E mutation gives resistance to long-tailed triazoles. Paradoxically the equivalent G73E mutation in ScErg11p gave increased susceptibility to all triazoles tested.
Conclusions: The bent conformation of ITC in the ScErg11p G73E mutant structure allowed interaction between the tail of the drug and the E73 carboxylate. This may explain increased susceptibility to ITC. The ScErg11p Y140F structure revealed normal binding to ITC, with the introduction of a water molecule at the mutation site. The wild type FLC structure showed polar interactions of a water with Y140, FLC and the carboxylate groups of the heme. The disruption or weakening of interactions between the drug and the enzyme may explain FLC resistance in the Y140F mutant. This study was supported by grants from the Marsden Fund and the HRCNZ.