Alanine scanning of transmembrane helix 11 of Cdr1p ABC antifungal efflux pump of Candida albicans: identification of amino acid residues critical for drug efflux.
OBJECTIVE
To investigate the role of transmembrane segment 11 (TMS11) of Candida albicans drug resistance protein (Cdr1p) in drug extrusion.


METHODS
We replaced each of the 21 putative residues of TMS11 with alanine by site-directed mutagenesis. The Saccharomyces cerevisiae AD1-8u(-) strain was used to overexpress the green fluorescent protein tagged wild-type and mutant variants of TMS11 of Cdr1p. The cells expressing mutant variants were functionally characterized.


RESULTS
Out of 21 residues of TMS11, substitution of seven residues, i.e. A1346G, A1347G, T1351A, T1355A, L1358A, F1360A and G1362A, affected differentially the substrate specificity of Cdr1p, while 14 mutants had no significant effect on Cdr1p function. TMS11 projection in an alpha-helical configuration revealed with few exceptions (A1346 and F1360), a distinct segregation of mutation-sensitive residues (A1347, T1351, T1355, L1358 and G1362) towards the more hydrophilic face. Interestingly, mutation-insensitive residues seem to cluster towards the hydrophobic side of the helix. Competition of rhodamine 6G efflux, in the presence of excess of various substrates in the cells expressing native Cdr1p, revealed for the first time the overlapping binding site between azoles (such as ketoconazole, miconazole and itraconazole) and rhodamine 6G. The ability of these azoles to compete with rhodamine 6G was completely lost in mutants F1360A and G1362A, while it was selectively lost in other variants of Cdr1p. We further confirmed that fungicidal synergism of calcineurin inhibitor FK520 with azoles is mediated by Cdr1p; wherein in addition to conserved T1351, substitution of T1355, L1358 and G1362 of TMS11 also resulted in abrogation of synergism.


CONCLUSIONS
Our study for the first time provides an insight into the possible role of TMS11 of Cdr1p in drug efflux.
Saini
Preeti
P
Membrane Biology Laboratory, School of Life Sciences, Jawaharlal Nehru University, New Delhi, India.
Prasad
Tulika
T
Gaur
Naseem Akhtar
NA
Shukla
Suneet
S
Jha
Sudhakar
S
Komath
Sneha Sudha
SS
Khan
Luqman Ahmad
LA
Haq
Qazi Mohd Rizwanul
QM
Prasad
Rajendra
R
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000935:Antifungal Agents / Q000493:pharmacokinetics*; D001483:Base Sequence; D001665:Binding Sites; D025141:Drug Resistance, Fungal; D005656:Fungal Proteins / Q000737:chemistry* / Q000502:physiology*; D007654:Ketoconazole / Q000493:pharmacokinetics; D026901:Membrane Transport Proteins / Q000737:chemistry* / Q000502:physiology*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017433:Protein Structure, Secondary; D012235:Rhodamines / Q000378:metabolism; D013379:Substrate Specificity
D000935:Antifungal Agents; C094693:CDR1 protein, Candida albicans; D005656:Fungal Proteins; D026901:Membrane Transport Proteins; D012235:Rhodamines; C026188:rhodamine 6G; D007654:Ketoconazole