Understanding the communication pathways between remote sites in proteins is of key importance for understanding their function and mechanism of action. These remain largely unexplored among the pleiotropic drug resistance (PDR) representatives of the ubiquitous superfamily of ATP-binding cassette (ABC) transporters. To identify functionally coupled residues important for the polyspecific transport by the fungal ABC multidrug transporter Cdr1p a new selection strategy, towards increased resistance to a preferred substrate of the homologous Snq2p, was applied to a library of randomly generated mutants. The single amino acid substitutions, located pseudosymmetrically in each domain of the internally duplicated protein: the H-loop of the N-terminal nucleotide binding domain (NBD1) (C363R) and in the C-terminal NBD2 region preceding Walker A (V885G). The central regions of the first transmembrane helices 1 and 7 of both transmembrane domains were also affected by the G521S/D and A1208V substitutions respectively. Although the mutants were expressed at a similar level and located correctly to the plasma membrane, they selectively affected transport of multiple drugs, including azole antifungals. The synergistic effects of combined mutations on drug resistance, drug dependent ATPase activity and transport support the view inferred from the statistical coupling analysis (SCA) of aminoacid coevolution and mutational analysis of other ABC transporter families that these residues are an important part of the conserved, allosterically coupled interdomain communication network. Our results shed new light on the communication between the pseudosymmetrically arranged domains in a fungal PDR ABC transporter and reveal its profound influence on substrate specificity.
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