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

ATPase activity of reconstituted LmrCD is stimulated by DARPin activators and daunomycin.Each symbol or bar represents the average of three data points. (A) The ATPase activity of reconstituted LmrCD is stimulated in the presence of daunomycin in a dose-dependent manner. (B) Reconstituted LmrCD (protein:lipid ratio of 1∶50, proteoliposomes diluted to obtain an LmrCD concentration of 70 nM) was incubated with DARPin activators and control DARPin E3_5* (2.5 µM) and the ATPase activity was determined in the absence and presence of 50 µM daunomycin (triplicates). As a control, buffer instead of DARPins were added to LmrCD. According to t-test analysis, the measured ATPase activity differences between DARPin_Act1 to Act3 and the buffer control are statistically significant (p<0.01 in the absence and p<0.05 in the presence of daunomycin, respectively). (C) The ATPase activities of LmrCD in the presence of DARPin_Act2 and E3_5 were determined over a range of ATP concentrations. The data points were fitted to the Hill equation.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3366976&req=5

pone-0037845-g009: ATPase activity of reconstituted LmrCD is stimulated by DARPin activators and daunomycin.Each symbol or bar represents the average of three data points. (A) The ATPase activity of reconstituted LmrCD is stimulated in the presence of daunomycin in a dose-dependent manner. (B) Reconstituted LmrCD (protein:lipid ratio of 1∶50, proteoliposomes diluted to obtain an LmrCD concentration of 70 nM) was incubated with DARPin activators and control DARPin E3_5* (2.5 µM) and the ATPase activity was determined in the absence and presence of 50 µM daunomycin (triplicates). As a control, buffer instead of DARPins were added to LmrCD. According to t-test analysis, the measured ATPase activity differences between DARPin_Act1 to Act3 and the buffer control are statistically significant (p<0.01 in the absence and p<0.05 in the presence of daunomycin, respectively). (C) The ATPase activities of LmrCD in the presence of DARPin_Act2 and E3_5 were determined over a range of ATP concentrations. The data points were fitted to the Hill equation.

Mentions: To further elucidate the mechanism by which the DARPin activators stimulate the function of LmrCD, detergent-purified LmrCD was reconstituted into proteoliposomes made of polar E.coli lipids and egg-phosphatidylcholine mixed at a ratio of 3∶1 [55]. Reconstituted LmrCD exhibits basal ATPase activities that are three times lower than the activity of purified LmrCD in its micellar form (not shown). Addition of increasing concentrations of daunomycin to reconstituted LmrCD (5–200 µM) increased its ATPase activity in a dose-dependent manner, reaching two-fold stimulation at 200 µM daunomycin (Figure 9A). The ATPase activity of reconstituted LmrCD in the presence of the DARPin activators and the control DARPin E3_5 was then compared to samples to which no DARPins were added (Figure 9B). The addition of DARPin E3_5 did not change the ATPase activity of LmrCD at any concentration of daunomycin. On the other hand, ATP hydrolysis of LmrCD was significantly stimulated upon addition of the three DARPin activators up to 1.6 fold in case of DARPin_Act2. These observations in proteoliposomes were found to be statistically significant in three independent reconstitution experiments, one of which is shown in Figure 9B. The DARPin activators are therefore capable of increasing the ATPase activity of LmrCD to a similar extent as 50 µM of daunomycin for which a 1.8 fold increase is seen (Figure 9A and B). The increase of LmrCD’s ATPase activity by the DARPin activators and daunomycin was found to be additive, suggesting that the molecular mechanism underlying these stimulatory effects are distinct. Basal and DARPin_Act2-stimulated ATPase activity of reconstituted LmrCD was further elucidated over a range of ATP concentrations (Figure 9C). The data was fitted using the Hill equation, and the apparent Km for ATP and Vmax of the ATPase reaction as well as the Hill coefficient were determined. The errors represent standard errors of the parameters derived from nonlinear regression analysis. In presence of DARPin_Act2, the apparent affinity of LmrCD for ATP was not significantly altered (Km,app of 0.85±0.06 mM and 0.73±0.09 mM for DARPin_Act2 and E3_5, respectively). Vmax on the other hand was doubled in the presence of DARPin_Act2 (Vmax of 500±22 nmol/min/mg of protein versus 247±19 nmol/min/mg of protein). The Hill coefficient was found to be unaltered in presence of DARPin_Act2 (2.0±0.3 and 2.0±0.5 for DARPin_Act2 and E3_5, respectively). The sigmoidal nature of the fitted curve suggests positive cooperativity between the non-canonical and the consensus composite catalytic site of LmrCD, a finding reminiscent of the maltose transporter and the isolated NBDs of HlyB [62], [63].


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)

ATPase activity of reconstituted LmrCD is stimulated by DARPin activators and daunomycin.Each symbol or bar represents the average of three data points. (A) The ATPase activity of reconstituted LmrCD is stimulated in the presence of daunomycin in a dose-dependent manner. (B) Reconstituted LmrCD (protein:lipid ratio of 1∶50, proteoliposomes diluted to obtain an LmrCD concentration of 70 nM) was incubated with DARPin activators and control DARPin E3_5* (2.5 µM) and the ATPase activity was determined in the absence and presence of 50 µM daunomycin (triplicates). As a control, buffer instead of DARPins were added to LmrCD. According to t-test analysis, the measured ATPase activity differences between DARPin_Act1 to Act3 and the buffer control are statistically significant (p<0.01 in the absence and p<0.05 in the presence of daunomycin, respectively). (C) The ATPase activities of LmrCD in the presence of DARPin_Act2 and E3_5 were determined over a range of ATP concentrations. The data points were fitted to the Hill equation.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0037845-g009: ATPase activity of reconstituted LmrCD is stimulated by DARPin activators and daunomycin.Each symbol or bar represents the average of three data points. (A) The ATPase activity of reconstituted LmrCD is stimulated in the presence of daunomycin in a dose-dependent manner. (B) Reconstituted LmrCD (protein:lipid ratio of 1∶50, proteoliposomes diluted to obtain an LmrCD concentration of 70 nM) was incubated with DARPin activators and control DARPin E3_5* (2.5 µM) and the ATPase activity was determined in the absence and presence of 50 µM daunomycin (triplicates). As a control, buffer instead of DARPins were added to LmrCD. According to t-test analysis, the measured ATPase activity differences between DARPin_Act1 to Act3 and the buffer control are statistically significant (p<0.01 in the absence and p<0.05 in the presence of daunomycin, respectively). (C) The ATPase activities of LmrCD in the presence of DARPin_Act2 and E3_5 were determined over a range of ATP concentrations. The data points were fitted to the Hill equation.
Mentions: To further elucidate the mechanism by which the DARPin activators stimulate the function of LmrCD, detergent-purified LmrCD was reconstituted into proteoliposomes made of polar E.coli lipids and egg-phosphatidylcholine mixed at a ratio of 3∶1 [55]. Reconstituted LmrCD exhibits basal ATPase activities that are three times lower than the activity of purified LmrCD in its micellar form (not shown). Addition of increasing concentrations of daunomycin to reconstituted LmrCD (5–200 µM) increased its ATPase activity in a dose-dependent manner, reaching two-fold stimulation at 200 µM daunomycin (Figure 9A). The ATPase activity of reconstituted LmrCD in the presence of the DARPin activators and the control DARPin E3_5 was then compared to samples to which no DARPins were added (Figure 9B). The addition of DARPin E3_5 did not change the ATPase activity of LmrCD at any concentration of daunomycin. On the other hand, ATP hydrolysis of LmrCD was significantly stimulated upon addition of the three DARPin activators up to 1.6 fold in case of DARPin_Act2. These observations in proteoliposomes were found to be statistically significant in three independent reconstitution experiments, one of which is shown in Figure 9B. The DARPin activators are therefore capable of increasing the ATPase activity of LmrCD to a similar extent as 50 µM of daunomycin for which a 1.8 fold increase is seen (Figure 9A and B). The increase of LmrCD’s ATPase activity by the DARPin activators and daunomycin was found to be additive, suggesting that the molecular mechanism underlying these stimulatory effects are distinct. Basal and DARPin_Act2-stimulated ATPase activity of reconstituted LmrCD was further elucidated over a range of ATP concentrations (Figure 9C). The data was fitted using the Hill equation, and the apparent Km for ATP and Vmax of the ATPase reaction as well as the Hill coefficient were determined. The errors represent standard errors of the parameters derived from nonlinear regression analysis. In presence of DARPin_Act2, the apparent affinity of LmrCD for ATP was not significantly altered (Km,app of 0.85±0.06 mM and 0.73±0.09 mM for DARPin_Act2 and E3_5, respectively). Vmax on the other hand was doubled in the presence of DARPin_Act2 (Vmax of 500±22 nmol/min/mg of protein versus 247±19 nmol/min/mg of protein). The Hill coefficient was found to be unaltered in presence of DARPin_Act2 (2.0±0.3 and 2.0±0.5 for DARPin_Act2 and E3_5, respectively). The sigmoidal nature of the fitted curve suggests positive cooperativity between the non-canonical and the consensus composite catalytic site of LmrCD, a finding reminiscent of the maltose transporter and the isolated NBDs of HlyB [62], [63].

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