Abstract
Cells control their volume through the accumulation of compatible solutes. The bacterial ATP-binding cassette transporter OpuA couples compatible solute uptake to ATP hydrolysis. Here, we study the gating mechanism and energy coupling of OpuA reconstituted in lipid nanodiscs. We show that anionic lipids are essential both for the gating and the energy coupling. The tight coupling between substrate binding on extracellular domains and ATP hydrolysis by cytoplasmic nucleotide-binding domains allows the study of transmembrane signaling in nanodiscs. From the tight coupling between processes at opposite sides of the membrane, we infer that the ATPase activity of OpuA in nanodiscs reflects solute translocation. Intriguingly, the substrate-dependent, ionic strength-gated ATPase activity of OpuA in nanodiscs is at least an order of magnitude higher than in lipid vesicles (i.e. with identical membrane lipid composition, ionic strength, and nucleotide and substrate concentrations). Even with the chemical components the same, the lateral pressure (profile) of the nanodiscs will differ from that of the vesicles. We thus propose that membrane tension limits translocation in vesicular systems. Increased macromolecular crowding does not activate OpuA but acts synergistically with ionic strength, presumably by favoring gating interactions of like-charged surfaces via excluded volume effects.
Highlights
We show strict coupling between glycine betaine binding and ATPase activity in OpuA nanodiscs, provided the chemical composition of the bilayer membrane and ionic strength of the medium are taken care of
In vitro, the number of ATP molecules hydrolyzed per substrate translocated of many ATP-binding cassette (ABC) transporters is very high and does not reflect the mechanistic stoichiometry and does not reflect translocation (44 – 45)
We note that the relatively high fraction of anionic lipids required for glycine betaine-dependent ATPase activity is physiologically relevant [46]
Summary
Significance: Tight coupling between substrate binding and ATP hydrolysis in ABC transporters allows the study of transmembrane signaling in nanodiscs. Cells control their volume through the accumulation of compatible solutes. The tight coupling between substrate binding on extracellular domains and ATP hydrolysis by cytoplasmic nucleotide-binding domains allows the study of transmembrane signaling in nanodiscs. Increased macromolecular crowding does not activate OpuA but acts synergistically with ionic strength, presumably by favoring gating interactions of likecharged surfaces via excluded volume effects. Less clear is the role of hydration and/or excluded volume (“crowding”) effects on the activation of these transporters These physicochemical parameters have been shown to influence the activity of ProP, either directly or via interactions of protein residues with the membrane [17, 18]. We used the OpuA nanodiscs to optimize the energy coupling and to determine the gating mechanism of the transporter
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
