Title: The Deviant ATP-binding Site of the Multidrug Efflux Pump Pdr5 Plays an Active Role in the Transport Cycle
Abstract: Pdr5 is the founding member of a large subfamily of evolutionarily distinct, clinically important fungal ABC transporters containing a characteristic, deviant ATP-binding site with altered Walker A, Walker B, Signature (C-loop), and Q-loop residues. In contrast to these motifs, the D-loops of the two ATP-binding sites have similar sequences, including a completely conserved aspartate residue. Alanine substitution mutants in the deviant Walker A and Signature motifs retain significant, albeit reduced, ATPase activity and drug resistance. The D-loop residue mutants D340A and D1042A showed a striking reduction in plasma membrane transporter levels. The D1042N mutation localized properly had nearly WT ATPase activity but was defective in transport and was profoundly hypersensitive to Pdr5 substrates. Therefore, there was a strong uncoupling of ATPase activity and drug efflux. Taken together, the properties of the mutants suggest an additional, critical intradomain signaling role for deviant ATP-binding sites.Background: The deviant ATP-binding site in the important drug resistance-linked Pdr subfamily is unique.Results: Mutations in conserved residues exhibit significant ATPase activity, but reduced transport activity.Conclusion: Conserved residues Cys-199, Glu-1013, and Asp-1042 are not directly involved in ATP hydrolysis but are actively involved in the transport cycle.Significance: Our results indicate a new role for deviant ATP-binding sites. Pdr5 is the founding member of a large subfamily of evolutionarily distinct, clinically important fungal ABC transporters containing a characteristic, deviant ATP-binding site with altered Walker A, Walker B, Signature (C-loop), and Q-loop residues. In contrast to these motifs, the D-loops of the two ATP-binding sites have similar sequences, including a completely conserved aspartate residue. Alanine substitution mutants in the deviant Walker A and Signature motifs retain significant, albeit reduced, ATPase activity and drug resistance. The D-loop residue mutants D340A and D1042A showed a striking reduction in plasma membrane transporter levels. The D1042N mutation localized properly had nearly WT ATPase activity but was defective in transport and was profoundly hypersensitive to Pdr5 substrates. Therefore, there was a strong uncoupling of ATPase activity and drug efflux. Taken together, the properties of the mutants suggest an additional, critical intradomain signaling role for deviant ATP-binding sites. Background: The deviant ATP-binding site in the important drug resistance-linked Pdr subfamily is unique. Results: Mutations in conserved residues exhibit significant ATPase activity, but reduced transport activity. Conclusion: Conserved residues Cys-199, Glu-1013, and Asp-1042 are not directly involved in ATP hydrolysis but are actively involved in the transport cycle. Significance: Our results indicate a new role for deviant ATP-binding sites.