Abstract
ABC transporters harness the energy from ATP binding and hydrolysis to translocate substrates across the membrane. Binding of two ATP molecules at the nucleotide binding domains (NBDs) leads to the formation of an outward-facing state. The conformational changes required to reset the transporter to the inward-facing state are initiated by sequential hydrolysis of the bound nucleotides. In a homodimeric ABC exporter such as MsbA responsible for lipid A transport in Escherichia coli, sequential ATP hydrolysis implies the existence of an asymmetric conformation. Here we report the in vitro selection of a designed ankyrin repeat protein (DARPin) specifically binding to detergent-solubilized MsbA. Only one DARPin binds to the homodimeric transporter in the absence as well as in the presence of nucleotides, suggesting that it recognizes asymmetries in MsbA. DARPin binding increases the rate of ATP hydrolysis by a factor of two independent of the substrate-induced ATPase stimulation. Electron paramagnetic resonance (EPR) measurements are found to be in good agreement with the available crystal structures and reveal that DARPin binding does not affect the large nucleotide-driven conformational changes of MsbA. The binding epitope was mapped by cross-linking and EPR to the membrane-spanning part of the transmembrane domain (TMD). Using cross-linked DARPin-MsbA complexes, 8-azido-ATP was found to preferentially photolabel one chain of the homodimer, suggesting that the asymmetries captured by DARPin binding at the TMDs are propagated to the NBDs. This work demonstrates that in vitro selected binders are useful tools to study the mechanism of membrane proteins.
Highlights
ATP-binding cassette (ABC) exporters are suggested to hydrolyze ATP sequentially implying the existence of asymmetries
From 2000 designed ankyrin repeat protein (DARPin) clones analyzed for binding to bMsbAAviC by an initial ELISA, 42 DARPins were further analyzed using a specificity ELISA in which besides bMsbAAviC the ABC transporter LmrCD and the secondary-active multidrug transporter AcrB were used
Using an in vitro selected DARPin binder, we provide here novel evidence supporting considerable asymmetries to be found in a well-studied ABC exporter
Summary
ABC exporters are suggested to hydrolyze ATP sequentially implying the existence of asymmetries. In a homodimeric ABC exporter such as MsbA responsible for lipid A transport in Escherichia coli, sequential ATP hydrolysis implies the existence of an asymmetric conformation. Using cross-linked DARPin-MsbA complexes, 8-azido-ATP was found to preferentially photolabel one chain of the homodimer, suggesting that the asymmetries captured by DARPin binding at the TMDs are propagated to the NBDs. This work demonstrates that in vitro selected binders are useful tools to study the mechanism of membrane proteins. Asymmetry in MsbA Recognized by a DARPin from 3.7 Å to 5.5 Å [9] Based on these crystal structures, large conformational changes are expected to occur in ABC exporters upon nucleotide binding and hydrolysis, which could be confirmed by cross-linking experiments and EPR measurements (10 –14). DARPin binding enhances the basal ATPase activity of the transporter without impairing the substrate-induced stimulation suggesting that the catalytic cycle is accelerated when asymmetric MsbA conformations are populated
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
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.
