Movement of the Nucleotide Binding Domains in the Reconstituted ABC Transporter MsbA During the ATP-Hydrolysis Cycle

Abstract

ATP-binding cassette (ABC) transporters move substrates across membranes, including nutrients, toxins, peptides and small inorganic ions. MsbA is a homodimeric bacterial lipid flippase homolog of P-glycoprotein, a transporter involved in multidrug resistance. ABC transporters are formed by two transmembrane domains and two highly conserved nucleotide binding domains (NBDs) that bind and hydrolyze ATP. Crystal structures have shown widely separated NBDs (open conformation; nucleotide-free state) and NBDs forming a dimer with nucleotide trapped at the interface (closed conformation, nucleotide-bound), leading to the proposal of a switch model, where NBDs associate/dissociate during the ATP hydrolysis cycle. Other proposed mechanisms suggest instead that the NBDs are always in contact. Recent studies using Luminescence Resonance Energy Transfer (LRET) in detergent-solubilized MsbA have shown transitions between completely separated and dimeric NBD, in agreement with the switch-model (Cooper & Altenberg, 2013. JBC 287:14994). Here, we used LRET to determine if complete NBD separation also occurs when MsbA is reconstituted in a membrane. Basically, the single cysteine MsbA (T561C) was labeled with LRET donor and acceptor probes, reconstituted in nanodiscs, and the donor-acceptor distance was determined at different steps during the ATP hydrolysis cycle. The reconstituted protein displayed higher ATPase activity than MsbA in detergent. Donor-acceptor distances in nucleotide-free and nucleotide-bound states, as well as during hydrolysis conditions (MgATP), indicated small distance changes, consistent with a partial opening of the NBD dimers. There was no evidence for the longer distance (>50 A) observed in the open conformation and in detergent. These results show a dramatic effect of the lipid bilayer on the molecular mechanism of MsbA, and suggest that the NBDs remain in close proximity during the hydrolysis cycle under more physiological conditions. This work was supported by CPRIT grant RP101073.