Structure and function analysis of CaMdr1p, a major facilitator superfamily antifungal efflux transporter protein of Candida albicans: identification of amino acid residues critical for drug/H+ transport.
We have cloned and overexpressed multidrug transporter CaMdr1p as a green fluorescent protein-tagged protein to show its capability to extrude drug substrates. The drug extrusion was sensitive to pH and energy inhibitors and displayed selective substrate specificity. CaMdr1p has a unique and conserved antiporter motif, also called motif C [G(X6)G(X3)GP(X2)GP(X2)G], in its transmembrane segment 5 (TMS 5). Alanine scanning of all the amino acids of the TMS 5 by site-directed mutagenesis highlighted the importance of the motif, as well as that of other residues of TMS 5, in drug transport. The mutant variants of TMS 5 were placed in four different categories. The first category had four residues, G244, G251, G255, and G259, which are part of the conserved motif C, and their substitution with alanine resulted in increased sensitivity to drugs and displayed impaired efflux of drugs. Interestingly, first category mutants, when replaced with leucine, resulted in more dramatic loss of drug resistance and efflux. Notwithstanding the location in the core motif, the second category included residues which are part of the motif, such as P260, and those which were not part of the motif, such as L245, W248, P256, and F262, whose substitution with alanine resulted in a severe loss of drug resistance and efflux. The third category included G263, which is a part of motif C, but unlike other conserved glycines, its replacement with alanine or leucine showed no change in the phenotype. The replacement of the remaining 11 residues of the fourth category did not result in any change. The putative helical wheel projection showed clustering of functionally critical residues to one side and thus suggests an asymmetric nature of TMS 5.
Pasrija
Ritu
R
Membrane Biology Lab, School of Life Sciences, Jawaharlal Nehru University, New Delhi 110067, India.
Banerjee
Dibyendu
D
Prasad
Rajendra
R
D018528:ATP-Binding Cassette Transporters / Q000737:chemistry*; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000187:drug effects / Q000235:genetics; D000935:Antifungal Agents / Q000494:pharmacology; D017920:Antiporters / Q000737:chemistry* / Q000378:metabolism; D001692:Biological Transport / Q000187:drug effects; D002176:Candida albicans / Q000187:drug effects / Q000378:metabolism*; D002352:Carrier Proteins / Q000502:physiology; D026141:Drug Resistance, Multiple, Fungal; D017136:Ion Transport; D026901:Membrane Transport Proteins / Q000737:chemistry* / Q000378:metabolism*; D008826:Microbial Sensitivity Tests; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000737:chemistry* / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding / Q000187:drug effects / Q000235:genetics; D013045:Species Specificity; D013329:Structure-Activity Relationship
D018528:ATP-Binding Cassette Transporters; D000935:Antifungal Agents; D017920:Antiporters; D002352:Carrier Proteins; D026901:Membrane Transport Proteins; D027425:ATP-Binding Cassette, Sub-Family C Proteins