Divergent signature motifs of nucleotide binding domains of ABC multidrug transporter, CaCdr1p of pathogenic Candida albicans, are functionally asymmetric and noninterchangeable

Abstract

Nucleotide binding domains (NBDs) of the multidrug transporter of Candida albicans, CaCdr1p, possess unique divergent amino acids in their conserved motifs. For example, NBD1 (N-terminal-NBD) possesses conserved signature motifs, while the same motif is divergent in NBD2 (C-terminal-NBD). In this study, we have evaluated the contribution of these conserved and divergent signature motifs of CaCdr1p in ATP catalysis and drug transport. By employing site-directed mutagenesis, we made three categories of mutant variants. These included mutants where all the signature motif residues were replaced with either alanines or mutants with exchanged equipositional residues to mimic the conservancy and degeneracy in opposite domain. In addition, a set of mutants where signature motifs were swapped to have variants with either both the conserved or degenerated entire signature motif. We observed that conserved and equipositional residues of NBD1 and NBD2 and swapped signature motif mutants showed high susceptibility to all the tested drugs with simultaneous abrogation in ATPase and R6G efflux activities. However, some of the mutants displayed a selective increase in susceptibility to the drugs. Notably, none of the mutant variants and WT-CaCdr1p showed any difference in drug and nucleotide binding. Our mutational analyses show not only that certain conserved residues of NBD1 signature sequence (S304, G306, and E307) are important in ATP hydrolysis and R6G efflux but also that a few divergent residues (N1002 and E1004) of NBD2 signature motif have evolved to be functionally relevant and are not interchangeable. Taken together, our data suggest that the signature motifs of CaCdr1p, whether it is divergent or conserved, are nonexchangeable and are functionally critical for ATP hydrolysis.

Fig. 1

Topology of CaCdr1p and sequence alignment of Signature motifs from various ABC transporters. The sequence alignment of Signature motif residues in NBDs with those from other nucleotide binding domains of some known ABC transporters is shown. Signature motifs of CaCdr1p are shown in bold and the conserved and divergent residues are underlined.

Fig. 2

Alanine scanning of the Signature motifs of CaCdr1p. (A) Fluorescence imaging (upper panel) by confocal microscope showing membrane localization of WT-CaCdr1p and its mutant variant protein expressing cells. Flow cytometry (lower panel) of S. cerevisiae cells expressing WT-CaCdr1p and its mutant variants. The histogram derived from the cell quest program depicts fluorescence intensities for AD1-8u- (control) (purple filled area) and WT-CaCdr1p (solid orange line) for each panel, and the other extra line represents that for the respective CaCdr1p mutant variant-expressing cells. B) Drug resistance profile of WT-CaCdr1p and its Signature motifs mutant variants were determined by spot assay. It was done as per protocol described earlier [34]. In the spot assays, 5 μl of five-fold serial dilutions of each yeast culture (each with cells suspended in normal saline to an OD of 0.1 (A₆₀₀) were spotted on YEPD plates in absence (control) and presence of the following drugs: FLC, 5 μg/mL; CYH, 0.15 μg/mL; ANI, 1.0 μg/mL; and R6G 5 μg/mL. C). R6G efflux (left Y-axis) indicated by blue diamond (◆) and oligomycin sensitive ATPase activity (right Y-axis) of WT-CaCdr1p with its mutant variants indicated by green square () [34, 37].

Fig. 3

Equipositional replacements of the Signature sequences of CaCdr1p. A). Fluorescence imaging (upper panel) by confocal microscope showing membrane localization of CaCdr1p and its mutant variant protein expressing cells. Flow cytometry (lower panel) of S. cerevisiae cells expressing WT-CaCdr1p and its mutant variants. The histogram derived from the cell quest program depicts fluorescence intensities for AD1-8u-(control) (purple filled area) and WT-CaCdr1p (solid orange line) for each panel, and the other extra line represents the respective CaCdr1p mutant variant-expressing cells. B). Drug resistance profile of WT-CaCdr1p and its Signature motif mutant variants was determined by spot assays as described for Fig 2. C). R6G efflux (left Y-axis) indicated by blue diamond (◆) and oligomycin sensitive ATPase activity (right Y-axis) of WT-CaCdr1p with its mutant variants indicated by green square () [34, 37].

Fig. 4

Swapping of Signature motifs of CaCdr1p. A) Schematic diagrams of different CaCdr1p Signature swapped mutants variants. The entire Signature motif of NBD1 (white)/NBD2 (purple) was mutated to generate constructs with two conserved (Signature 1-1), two degenerated (Signature 2-2) and swapped (Signature 2-1) active site (upper panel). Localization and expression profile of WT-CaCdr1p and its swapped Signature mutant variants (Lower panel). B) Drug resistance profile of WT-CaCdr1p and its Signature motifs swapped mutants were determined by spot assays. C) Comparison of oligomycin sensitive ATPase activity of WT-CaCdr1p with its swapped mutant variants. ATPase activity of the PM fraction of cells expressing the WT-CaCdr1p and its mutant variants were assayed as described earlier [34]. D) R6G efflux by the WT-CaCdr1p and its mutant variant protein-expressing cells. The R6G efflux was measured as described previously [37]. The values are mean SD (± error bars) for three independent experiments.

Fig. 5

A). (i) Photoaffinity labeling of WT-CaCdr1p and its mutant variants with [¹²⁵I]-IAAP. The PM fraction (30 μg protein) of cells expressing WT-CaCdr1p and its mutant variants were incubated with 7.5 nM [¹²⁵I]-IAAP (2300 Ci/mmol). The samples were UV crosslinked and processed as described elsewhere [34]. (ii) Western blot analysis using anti-GFP antibody to ensure equal loading of WT-CaCdr1p and its Signature mutant variants (iii) Normalized incorporated [¹²⁵I] IAAP labeling with Western blot intensity. The values are shown in percentage. B). Photoaffinity labeling of WT-CaCdr1p and its mutant variants with [α-³²P] 8-azido ATP. The PM fraction (30 μg) of cells expressing the WT-CaCdr1p and its mutant variants were incubated with 10 μM [α-³²P] 8-azido ATP 7.5 μCi/nmol at 4°C and competed with 10 mM cold ATP (+ATP lane) as described in [34]. (ii) Western blot analysis using anti-GFP antibody to ensure equal loading of WT-CaCdr1p and its Signature mutant variants (iii) Normalized incorporated [α-³²P] 8-azido ATP labeling with Western blot intensity. The values are shown in percentage.

Fig. 5

A). (i) Photoaffinity labeling of WT-CaCdr1p and its mutant variants with [¹²⁵I]-IAAP. The PM fraction (30 μg protein) of cells expressing WT-CaCdr1p and its mutant variants were incubated with 7.5 nM [¹²⁵I]-IAAP (2300 Ci/mmol). The samples were UV crosslinked and processed as described elsewhere [34]. (ii) Western blot analysis using anti-GFP antibody to ensure equal loading of WT-CaCdr1p and its Signature mutant variants (iii) Normalized incorporated [¹²⁵I] IAAP labeling with Western blot intensity. The values are shown in percentage. B). Photoaffinity labeling of WT-CaCdr1p and its mutant variants with [α-³²P] 8-azido ATP. The PM fraction (30 μg) of cells expressing the WT-CaCdr1p and its mutant variants were incubated with 10 μM [α-³²P] 8-azido ATP 7.5 μCi/nmol at 4°C and competed with 10 mM cold ATP (+ATP lane) as described in [34]. (ii) Western blot analysis using anti-GFP antibody to ensure equal loading of WT-CaCdr1p and its Signature mutant variants (iii) Normalized incorporated [α-³²P] 8-azido ATP labeling with Western blot intensity. The values are shown in percentage.