title: Mutational Analysis of Intracellular Loops Identify Cross Talk with Nucleotide Binding Domains of Yeast ABC Transporter Cdr1p.
abstract: The ABC transporter Cdr1 protein (Cdr1p) of Candida albicans, which plays a major role in antifungal resistance, has two transmembrane domains (TMDs) and two nucleotide binding domains (NBDs) that are interconnected by extracellular (ECLs) and intracellular (ICLs) loops. To examine the communication interface between the NBDs and ICLs of Cdr1p, we subjected all four ICLs to alanine scanning mutagenesis, replacing each of the 85 residues with an alanine. The resulting ICL mutant library was analyzed by biochemical and phenotypic mapping. Only 18% of the mutants from this library displayed enhanced drug susceptibility. Most of the drug-susceptible mutants displayed uncoupling between ATP hydrolysis and drug transport. The two drug-susceptible ICL1 mutants (I574A and S593A) that lay within or close to the predicted coupling helix yielded two chromosomal suppressor mutations that fall near the Q-loop of NBD2 (R935) and in the Walker A motif (G190) of NBD1. Based on a 3D homology model and kinetic analysis of drug transport, our data suggest that large distances between ICL residues and their respective chromosomal suppressor mutations rule out a direct interaction between them. However, they impact the transport cycle by restoring the coupling interface via indirect downstream signaling.
authors.affiliation: School of Life Sciences, Jawaharlal Nehru University, New Delhi 110067, India.
authors.forename: Abdul Haseeb
authors.initials: AH
authors.lastname: Shah
authors.affiliation: School of Life Sciences, Jawaharlal Nehru University, New Delhi 110067, India.
authors.forename: Manpreet Kaur
authors.initials: MK
authors.lastname: Rawal
authors.affiliation: School of Life Sciences, Jawaharlal Nehru University, New Delhi 110067, India.
authors.forename: Sanjiveeni
authors.initials: S
authors.lastname: Dhamgaye
authors.affiliation: School of Life Sciences, Jawaharlal Nehru University, New Delhi 110067, India.
authors.forename: Sneha Sudha
authors.initials: SS
authors.lastname: Komath
authors.affiliation: School of Life Sciences, Jawaharlal Nehru University, New Delhi 110067, India.
authors.forename: Ajay Kumar
authors.initials: AK
authors.lastname: Saxena
authors.affiliation: School of Life Sciences, Jawaharlal Nehru University, New Delhi 110067, India.
authors.forename: Rajendra
authors.initials: R
authors.lastname: Prasad
mesh_terms: D018528:ATP-Binding Cassette Transporters / Q000235:genetics*; D019943:Amino Acid Substitution / Q000235:genetics; D000935:Antifungal Agents / Q000494:pharmacology*; D001393:Azoles / Q000494:pharmacology; D001692:Biological Transport / Q000235:genetics; D002176:Candida albicans / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism; D004252:DNA Mutational Analysis; D004268:DNA-Binding Proteins / Q000235:genetics; D025141:Drug Resistance, Fungal / Q000235:genetics*; D005656:Fungal Proteins / Q000235:genetics*; D026901:Membrane Transport Proteins / Q000235:genetics*; D008826:Microbial Sensitivity Tests; D017434:Protein Structure, Tertiary
chemical_list: D018528:ATP-Binding Cassette Transporters; D000935:Antifungal Agents; D001393:Azoles; C094693:CDR1 protein, Candida albicans; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; D026901:Membrane Transport Proteins