S279 point mutations in Candida albicans Sterol 14-α demethylase (CYP51) reduce in vitro inhibition by fluconazole

Abstract

The effects of S279F and S279Y point mutations in Candida albicans CYP51 (CaCYP51) on protein activity and on substrate (lanosterol) and azole antifungal binding were investigated. Both S279F and S279Y mutants bound lanosterol with 2-fold increased affinities (K(s), 7.1 and 8.0 μM, respectively) compared to the wild-type CaCYP51 protein (K(s), 13.5 μM). The S279F and S279Y mutants and the wild-type CaCYP51 protein bound fluconazole, voriconazole, and itraconazole tightly, producing typical type II binding spectra. However, the S279F and S279Y mutants had 4- to 5-fold lower affinities for fluconazole, 3.5-fold lower affinities for voriconazole, and 3.5- to 4-fold lower affinities for itraconazole than the wild-type CaCYP51 protein. The S279F and S279Y mutants gave 2.3- and 2.8-fold higher 50% inhibitory concentrations (IC₅₀s) for fluconazole in a CYP51 reconstitution assay than the wild-type protein did. The increased fluconazole resistance conferred by the S279F and S279Y point mutations appeared to be mediated through a combination of a higher affinity for substrate and a lower affinity for fluconazole. In addition, lanosterol displaced fluconazole from the S279F and S279Y mutants but not from the wild-type protein. Molecular modeling of the wild-type protein indicated that the oxygen atom of S507 interacts with the second triazole ring of fluconazole, assisting in orientating fluconazole so that a more favorable binding conformation to heme is achieved. In contrast, in the two S279 mutant proteins, this S507-fluconazole interaction is absent, providing an explanation for the higher K(d) values observed.

Fig 1

Alignment of Candida species and fungal CYP51 amino acid sequences. (A) CYP51 amino acid sequences of the Candida species Candida albicans (UniProtKB accession number P10613), Candida dubliniensis (Q96VP2), Candida tropicalis (P14263), Candida parapsilosis (C7EXA5), and Candida glabrata (P50859) were aligned using ClustalX. (B) Similarly, CYP51 amino acid sequences of Candida albicans CYP51 (P10613) and 10 other fungal CYP51 proteins, including those from Mycosphaerella graminicola (Q5XWE5), Botryotinia fuckeliana (Q9P428), Aspergillus fumigatus (CYP51B) (Q96W81), Penicillium digitatum (Q9P340), Aspergillus fumigatus (CYP51A) (Q4WNT5), Schizosaccharomyces pombe (Q09736), Phanerochaete chrysosporium (B6DX27), Cryptococcus neoformans (AAF35366), Ustilago maydis (P49602), and Saccharomyces cerevisiae (P10614), were aligned using ClustalX. Alignments of amino acid sequences surrounding the S279 residue of Candida albicans CYP51 are shown with numbering relating to the amino acid positions of Candida albicans CYP51 (P10613). Conserved residues are indicated by asterisks.

Fig 2

Absolute spectra of CaCYP51 proteins. The absolute spectra of 5 μM wild-type (solid lines), S279F (dashed lines), and S279Y (dotted lines) CaCYP51 proteins were determined between 300 and 700 nm under oxidative conditions in the absence (A) and presence (B) of 6 μM fluconazole. Absolute spectra were also recorded under reductive conditions (10 mM sodium dithionite) in the absence (C) and presence (D) of carbon monoxide.

Fig 3

Lanosterol binding properties of CaCYP51 proteins. Type I difference spectra were obtained by progressive titration of lanosterol against 5 μM wild-type (A), S279F (B), and S279Y (C) CaCYP51 proteins. (D) Lanosterol saturation curves were constructed using the Hill equation for the wild-type (filled circles), S279F (hollow circles), and S279Y (bulleted circles) CaCYP51 proteins.

Fig 4

Fluconazole binding properties of CaCYP51 proteins. Type II difference spectra were obtained for 5 μM wild-type (A), S279F (B), and S279Y (C) CaCYP51 proteins by progressive titration with fluconazole. (D) Fluconazole binding saturation curves were constructed using a rearrangement of the Morrison equation (26) for the wild-type (filled circles), S279F (hollow circles), and S279Y (bulleted circles) CaCYP51 proteins. Experiments were performed in triplicate, although data for only one replicate are shown.

Fig 5

Displacement of bound lanosterol from CaCYP51 proteins by fluconazole. Aqueous lanosterol (80 μM) was mixed with 5 μM CaCYP51 for 5 min. Bound lanosterol was then displaced by progressive titration with fluconazole and monitored spectrophotometrically between 350 and 500 nm for the wild-type (A), S279F (B), and S279Y (C) CaCYP51 proteins. (D) Fluconazole binding saturation curves were constructed using a rearrangement of the Morrison equation (26) for the wild-type protein (filled circles) and using the Michaelis-Menten equation for the S279F (hollow circles) and S279Y (bulleted circles) CaCYP51 proteins, where fluconazole binding was no longer tight. Experiments were performed in triplicate, although data for only one replicate are shown.

Fig 6

Determination of IC₅₀ and ΔA₅₀ values for fluconazole. (A) CYP51 reconstitution assays with 2.5 μM wild-type (filled circles), S279F (hollow circles), and S279Y (bulleted circles) CaCYP51 proteins were performed as detailed in Materials and Methods. The fluconazole concentration was varied from 0 to 4 μM, with the DMSO concentration kept constant at 0.5% (vol/vol). Mean enzyme velocities (v) for three replicates are shown along with associated standard error bars. (B) Fluconazole displacement by lanosterol was determined by incubating 5 μM CaCYP51 with fluconazole (0 to 12 μM) for 5 min prior to addition of 80 μM lanosterol and measuring the subsequent type I binding spectra for wild-type (filled circles), S279F (hollow circles), and S279Y (bulleted circles) CaCYP51 proteins. ΔA values were derived from the type I binding spectra obtained.

Fig 7

Structural modeling of conformational differences in wild-type, S279F, and S279Y CaCYP51 proteins. (A) Wild-type protein, with S279 located peripherally on an external β turn. The S279F (B) and S279Y (C) mutant proteins are also shown, with F279 and Y279 incorporated within a short section of α helix preceding the long I helix.

Fig 8

Structural modeling of fluconazole binding to wild-type, S279F, and S279Y CaCYP51 proteins. The wild-type protein shows fluconazole in the center (colored by element) and the heme group to the right, and residues predicted to be within the range of contact of fluconazole are labeled. The S279F and S279Y mutant proteins are also shown, with both S279 substitutions leading to conformational changes that result in S507 being removed from interaction with fluconazole but still bordering the heme cavity.