Abstract
ATP-binding cassette (ABC) transporters are found in all forms of life from microbes to humans, and transport a wide variety of substrates across the cell membrane using the energy released from ATP hydrolysis and an alternating-access mechanism. MsbA is a homodimeric ABC exporter from Gram-negative bacteria, and transports amphipathic substrates including precursors of lipopolysaccharides from the inner leaflet to the outer leaflet of the cytoplasmic membrane. Despite extensive structural and functional studies, controversies remain regarding the dynamic properties of the conformational changes of MsbA during its transport cycle in the lipid environment. Here, we used single-molecule fluorescence resonance energy transfer (smFRET) to explore the dynamic behaviors of MsbA in detergent micelles, nanodiscs, and proteoliposomes. MsbA reconstituted into liposomes showed higher transition frequency between different states on the cytoplasmic side, whereas detergent-solubilized MsbA showed higher transition frequency on the periplasmic side. Three major states were identified from this smFRET study in the functional cycle of MsbA, including an intermediate conformation between the fully opened and fully closed cytoplasmic conformations, associated with both ATP binding and hydrolysis.
Highlights
Adenosine triphosphate (ATP)-binding cassette (ABC) transporters are a large superfamily of membrane proteins that actively translocate a wide variety of substrates across the cellular membrane, driven by energy released from ATP hydrolysis (Dassa 2011; Davidson and Chen 2004; Higgins 2007; Locher 2016; Zhang et al 2016)
Experimental design for single-molecule fluorescence resonance energy transfer (smFRET) analysis smFRET experiments were performed on variants of Escherichia coli MsbA containing a single cysteine (Cys) residue in each subunit linked to fluorophores by maleimide chemistry
To better mimic the membrane environment, we further performed smFRET experiments with MsbA proteins reconstituted into either liposomes or nanodiscs, both of which were made from E. coli total lipids, and the reconstituted proteins were surface immobilized through biotin-labeled lipids to streptavidin-decorated channels (Fig. 1B)
Summary
Adenosine triphosphate (ATP)-binding cassette (ABC) transporters are a large superfamily of membrane proteins that actively translocate a wide variety of substrates across the cellular membrane, driven by energy released from ATP hydrolysis (Dassa 2011; Davidson and Chen 2004; Higgins 2007; Locher 2016; Zhang et al 2016). Architecture containing two transmembrane domains (TMDs) and two cytoplasmic nucleotide-binding domains (NBDs). The TMDs, which show high sequence diversity, form the translocation pathway and assume the role of substrate selectivity, whereas the NBDs are highly conserved and contain the ABC-characteristic motifs that bind ATP and catalyze its hydrolysis (Ward et al 2007). ABC transporters are classified into importers and exporters depending on the direction of substrate translocation (Zhang et al 2016). MsbA from Gram-negative bacteria is an extensively studied ABC exporter (Doerrler and Raetz 2002). This 128-kDa homodimeric transporter is located in the cytoplasmic (inner) membrane and plays a critical role in flipping Lipid A, a precursor of lipopolysaccharides, a
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