Limits...
Tuning the drug efflux activity of an ABC transporter in vivo by in vitro selected DARPin binders.

Seeger MA, Mittal A, Velamakanni S, Hohl M, Schauer S, Salaa I, Grütter MG, van Veen HW - PLoS ONE (2012)

Bottom Line: Consistent with this, purified activator DARPins were found to stimulate the ATPase activity of LmrCD in vitro when reconstituted in proteoliposomes.This study suggests that membrane transporters are tunable in vivo by in vitro selected binding proteins.Our approach could be of biopharmaceutical importance and might facilitate studies on molecular mechanisms of ABC transporters.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, University of Cambridge, Cambridge, United Kingdom. m.seeger@bioc.uzh.ch

ABSTRACT
ABC transporters use the energy from binding and hydrolysis of ATP to import or extrude substrates across the membrane. Using ribosome display, we raised designed ankyrin repeat proteins (DARPins) against detergent solubilized LmrCD, a heterodimeric multidrug ABC exporter from Lactococcus lactis. Several target-specific DARPin binders were identified that bind to at least three distinct, partially overlapping epitopes on LmrD in detergent solution as well as in native membranes. Remarkably, functional screening of the LmrCD-specific DARPin pools in L. lactis revealed three homologous DARPins which, when generated in LmrCD-expressing cells, strongly activated LmrCD-mediated drug transport. As LmrCD expression in the cell membrane was unaltered upon the co-expression of activator DARPins, the activation is suggested to occur at the level of LmrCD activity. Consistent with this, purified activator DARPins were found to stimulate the ATPase activity of LmrCD in vitro when reconstituted in proteoliposomes. This study suggests that membrane transporters are tunable in vivo by in vitro selected binding proteins. Our approach could be of biopharmaceutical importance and might facilitate studies on molecular mechanisms of ABC transporters.

Show MeSH

Related in: MedlinePlus

DARPin binding to membrane-embedded LmrCD.(A) Six DARPins (each at a 350 nM concentration) specific for AcrB or LmrCD were probed for binding to ISOVs containing either overproduced AcrBAviC or LmrCDAviC. Bound DARPins were detected on Western blot (left panel). The signals of the DARPin-specific bands were quantified by densitometry (right panel). Total binding denotes the quantified amount of DARPin bound to membrane vesicles containing overexpressed target protein. Background binding refers to binding to membrane vesicles containing overexpressed LmrCDAviC in case of the AcrB DARPin 110819, or overexpressed AcrBAviC when LmrCD-specific DARPins were used. Specific binding was calculated by subtracting background binding from total binding. (B) Binding of DARPin_Act2 and α-LmrCD#3 to ISOVs containing either overproduced AcrBAviC or LmrCDAviC was further assessed using increasing concentrations of DARPin (0.35 µM, 1 µM and 2 µM) and analyzed by Western blot (left panel). The data was quantified as in (A) (right panel). The data represent typical results observed in n = 3 experiments.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3366976&req=5

pone-0037845-g008: DARPin binding to membrane-embedded LmrCD.(A) Six DARPins (each at a 350 nM concentration) specific for AcrB or LmrCD were probed for binding to ISOVs containing either overproduced AcrBAviC or LmrCDAviC. Bound DARPins were detected on Western blot (left panel). The signals of the DARPin-specific bands were quantified by densitometry (right panel). Total binding denotes the quantified amount of DARPin bound to membrane vesicles containing overexpressed target protein. Background binding refers to binding to membrane vesicles containing overexpressed LmrCDAviC in case of the AcrB DARPin 110819, or overexpressed AcrBAviC when LmrCD-specific DARPins were used. Specific binding was calculated by subtracting background binding from total binding. (B) Binding of DARPin_Act2 and α-LmrCD#3 to ISOVs containing either overproduced AcrBAviC or LmrCDAviC was further assessed using increasing concentrations of DARPin (0.35 µM, 1 µM and 2 µM) and analyzed by Western blot (left panel). The data was quantified as in (A) (right panel). The data represent typical results observed in n = 3 experiments.

Mentions: The binding of DARPins to inside-out membrane vesicles (ISOVs) containing either overproduced AcrBAviC or LmrCDAviC was further characterized (Figure 8). Based on an analysis using a protease-cleavable LmrCD-GFP construct (see Materials and Methods), ISOV preparations were found to contain up to 10% of the membrane vesicles in the right-side-out orientation (right-side-out membrane vesicles, RSOVs). Total binding was determined as the amount of DARPin bound to ISOVs containing the overexpressed target protein. Background binding refers to binding of the respective DARPin to ISOVs containing an overexpressed membrane protein that is not recognized by the binder. For the AcrB-specific DARPin 110819, the membrane vesicles used for the determination of background binding thus contained overexpressed LmrCD and vice versa. Specific binding was then calculated by subtracting background binding from total binding. Binding of all six DARPins tested was target-specific, meaning that total binding was stronger than background binding. The AcrB-specific DARPin 110819, whose structure has been solved in complex with AcrB by X-ray crystallography, was used as control. As expected, DARPin 110819 binds relatively poorly to ISOVs despite its high reported binding affinity of 28 nM because the binding epitope on AcrB is located at the periplasmic loops and is therefore predominantly hidden in the vesicle lumen [18]. The binding signal for DARPin 110819 therefore originates from the estimated 10% RSOVs present in the ISOV preparation. Despite the fact that AcrB is expressed better than LmrCD (not shown), binding of α-LmrCD#2 and DARPin_Act3 to LmrCD-containing ISOVs resulted in signals that were around three times bigger than the ones of DARPin 110819 binding to AcrB-containing ISOVs (Figure 8A). Since the binding affinities of α-LmrCD#2 (9 nM) and DARPin_Act3 (55 nM) are in the same order of magnitude as of DARPin 110819 (28 nM), these LmrCD-specific DARPins appear to recognize epitopes at the cytoplasmic portion of LmrD, which are accessible in ISOVs. Specific binding of α-LmrCD#1 on the other hand is half as high as for DARPin 110819 whereas it is roughly the same for α-LmrCD#3. DARPin binding to these epitopes is therefore either restricted in membrane-embedded LmrCD or the epitope is only accessible from the physiological outside of the membrane. We also attempted to perform these DARPin binding experiments using RSOVs generated from E. coli using the EDTA-lysozyme method [60]. Studies on the accessibility of a C-terminal GFP fusion partner on LmrD to protease cleavage from the external surface of membrane vesicles indicated that, despite careful preparations, a substantial portion (up to 50%) of LmrCD-GFP containing membrane vesicles were in the inside-out orientation, and that therefore, this type of membrane vesicles could not be used to study the accessibility of the binding epitopes (data not shown). Background binding to ISOVs varied between the different DARPins and correlated with the aggregation behavior on SEC (Figure S1). Low background binding was observed for the DARPins α-LmrCD#1, α-LmrCD#3 and the AcrB-DARPin 110819, whereas α-LmrCD#2, DARPin_Act2 and DARPin_Act3 interacted with membrane vesicles lacking the target protein (Figure 8A).


Tuning the drug efflux activity of an ABC transporter in vivo by in vitro selected DARPin binders.

Seeger MA, Mittal A, Velamakanni S, Hohl M, Schauer S, Salaa I, Grütter MG, van Veen HW - PLoS ONE (2012)

DARPin binding to membrane-embedded LmrCD.(A) Six DARPins (each at a 350 nM concentration) specific for AcrB or LmrCD were probed for binding to ISOVs containing either overproduced AcrBAviC or LmrCDAviC. Bound DARPins were detected on Western blot (left panel). The signals of the DARPin-specific bands were quantified by densitometry (right panel). Total binding denotes the quantified amount of DARPin bound to membrane vesicles containing overexpressed target protein. Background binding refers to binding to membrane vesicles containing overexpressed LmrCDAviC in case of the AcrB DARPin 110819, or overexpressed AcrBAviC when LmrCD-specific DARPins were used. Specific binding was calculated by subtracting background binding from total binding. (B) Binding of DARPin_Act2 and α-LmrCD#3 to ISOVs containing either overproduced AcrBAviC or LmrCDAviC was further assessed using increasing concentrations of DARPin (0.35 µM, 1 µM and 2 µM) and analyzed by Western blot (left panel). The data was quantified as in (A) (right panel). The data represent typical results observed in n = 3 experiments.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3366976&req=5

pone-0037845-g008: DARPin binding to membrane-embedded LmrCD.(A) Six DARPins (each at a 350 nM concentration) specific for AcrB or LmrCD were probed for binding to ISOVs containing either overproduced AcrBAviC or LmrCDAviC. Bound DARPins were detected on Western blot (left panel). The signals of the DARPin-specific bands were quantified by densitometry (right panel). Total binding denotes the quantified amount of DARPin bound to membrane vesicles containing overexpressed target protein. Background binding refers to binding to membrane vesicles containing overexpressed LmrCDAviC in case of the AcrB DARPin 110819, or overexpressed AcrBAviC when LmrCD-specific DARPins were used. Specific binding was calculated by subtracting background binding from total binding. (B) Binding of DARPin_Act2 and α-LmrCD#3 to ISOVs containing either overproduced AcrBAviC or LmrCDAviC was further assessed using increasing concentrations of DARPin (0.35 µM, 1 µM and 2 µM) and analyzed by Western blot (left panel). The data was quantified as in (A) (right panel). The data represent typical results observed in n = 3 experiments.
Mentions: The binding of DARPins to inside-out membrane vesicles (ISOVs) containing either overproduced AcrBAviC or LmrCDAviC was further characterized (Figure 8). Based on an analysis using a protease-cleavable LmrCD-GFP construct (see Materials and Methods), ISOV preparations were found to contain up to 10% of the membrane vesicles in the right-side-out orientation (right-side-out membrane vesicles, RSOVs). Total binding was determined as the amount of DARPin bound to ISOVs containing the overexpressed target protein. Background binding refers to binding of the respective DARPin to ISOVs containing an overexpressed membrane protein that is not recognized by the binder. For the AcrB-specific DARPin 110819, the membrane vesicles used for the determination of background binding thus contained overexpressed LmrCD and vice versa. Specific binding was then calculated by subtracting background binding from total binding. Binding of all six DARPins tested was target-specific, meaning that total binding was stronger than background binding. The AcrB-specific DARPin 110819, whose structure has been solved in complex with AcrB by X-ray crystallography, was used as control. As expected, DARPin 110819 binds relatively poorly to ISOVs despite its high reported binding affinity of 28 nM because the binding epitope on AcrB is located at the periplasmic loops and is therefore predominantly hidden in the vesicle lumen [18]. The binding signal for DARPin 110819 therefore originates from the estimated 10% RSOVs present in the ISOV preparation. Despite the fact that AcrB is expressed better than LmrCD (not shown), binding of α-LmrCD#2 and DARPin_Act3 to LmrCD-containing ISOVs resulted in signals that were around three times bigger than the ones of DARPin 110819 binding to AcrB-containing ISOVs (Figure 8A). Since the binding affinities of α-LmrCD#2 (9 nM) and DARPin_Act3 (55 nM) are in the same order of magnitude as of DARPin 110819 (28 nM), these LmrCD-specific DARPins appear to recognize epitopes at the cytoplasmic portion of LmrD, which are accessible in ISOVs. Specific binding of α-LmrCD#1 on the other hand is half as high as for DARPin 110819 whereas it is roughly the same for α-LmrCD#3. DARPin binding to these epitopes is therefore either restricted in membrane-embedded LmrCD or the epitope is only accessible from the physiological outside of the membrane. We also attempted to perform these DARPin binding experiments using RSOVs generated from E. coli using the EDTA-lysozyme method [60]. Studies on the accessibility of a C-terminal GFP fusion partner on LmrD to protease cleavage from the external surface of membrane vesicles indicated that, despite careful preparations, a substantial portion (up to 50%) of LmrCD-GFP containing membrane vesicles were in the inside-out orientation, and that therefore, this type of membrane vesicles could not be used to study the accessibility of the binding epitopes (data not shown). Background binding to ISOVs varied between the different DARPins and correlated with the aggregation behavior on SEC (Figure S1). Low background binding was observed for the DARPins α-LmrCD#1, α-LmrCD#3 and the AcrB-DARPin 110819, whereas α-LmrCD#2, DARPin_Act2 and DARPin_Act3 interacted with membrane vesicles lacking the target protein (Figure 8A).

Bottom Line: Consistent with this, purified activator DARPins were found to stimulate the ATPase activity of LmrCD in vitro when reconstituted in proteoliposomes.This study suggests that membrane transporters are tunable in vivo by in vitro selected binding proteins.Our approach could be of biopharmaceutical importance and might facilitate studies on molecular mechanisms of ABC transporters.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, University of Cambridge, Cambridge, United Kingdom. m.seeger@bioc.uzh.ch

ABSTRACT
ABC transporters use the energy from binding and hydrolysis of ATP to import or extrude substrates across the membrane. Using ribosome display, we raised designed ankyrin repeat proteins (DARPins) against detergent solubilized LmrCD, a heterodimeric multidrug ABC exporter from Lactococcus lactis. Several target-specific DARPin binders were identified that bind to at least three distinct, partially overlapping epitopes on LmrD in detergent solution as well as in native membranes. Remarkably, functional screening of the LmrCD-specific DARPin pools in L. lactis revealed three homologous DARPins which, when generated in LmrCD-expressing cells, strongly activated LmrCD-mediated drug transport. As LmrCD expression in the cell membrane was unaltered upon the co-expression of activator DARPins, the activation is suggested to occur at the level of LmrCD activity. Consistent with this, purified activator DARPins were found to stimulate the ATPase activity of LmrCD in vitro when reconstituted in proteoliposomes. This study suggests that membrane transporters are tunable in vivo by in vitro selected binding proteins. Our approach could be of biopharmaceutical importance and might facilitate studies on molecular mechanisms of ABC transporters.

Show MeSH
Related in: MedlinePlus