Genetic and Biochemical Characterization of the Signaling Interface and the Deviant ATP Binding Site in the Yeast Multidrug Transporter Pdr5
Pdr5 is an ABC transporter in Saccharomyces cerevisiae that facilitates xenobiotic efflux. Like all ABC transporters Pdr5 is composed of a pair of transmembrane domain (TMDs) that contain substrate binding pockets and a pair of nucleotide binding domains (NBDs) that power substrate transport. The NBD contains conserved motifs including Walker A, Walker B, Q-loop and Signature that facilitate ATP binding/hydrolysis. At each ATP binding site, an ATP molecule is sandwiched between Walker A, Walker B, Q-loop of one NBD and Signature of the other. In the Pdr family of fungal transporters, one of the ATP binding sites is canonical, known to bind/hydrolyze ATP. The other ATP binding site, however, is made up deviant residues.Hydrolysis of ATP at the NBD is thought to cause a conformational change at the TMDs. X-ray structure of Sav1866 implicates Q-loop and the intercellular loops in communication between the TMDs and the NBDs. In order to determine the signal interface in Pdr5, we evaluated the role of the Q loop residues, E244 in NBD1 and Q951 in NBD2. Surprisingly, mutation of these conserved residues exhibited only mild drug hyper sensitivity and retained significant ATPase activity. The double mutant showed a greater than additive sensitivity, indicating a functional overlap between the two Q loops. Interestingly, the reduced ATPase activity of the double mutant was equalto the single mutants. It is likely, therefore that the Q-loop residues are involved in interdomain communication.The precise function of the deviant ATP site in Pdr5 remains unclear. In order to elucidate its role, we evaluated the contribution of the deviant Walker A (C199) and a residue in the deviant signature motif (E1013). Mutating these residues to alanine exhibited only moderate drug hypersensitivity. They also retained significant ATPase activity, indicating that the deviant Walker A and Signature are not catalytic. As expected, mutating K911 in Walker A and G312 in the signature of the canonical site creates a null phenotype. This indicates that the deviant and the canonical ATP binding sites are not equivalent. We propose that the deviant site might regulate ATP hydrolysis at the canonical site.
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