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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
Ribosome display and ELISA set-up.(A) Sketch delineating one DARPin selection round using ribosome display (adopted from [31]). The DARPin library in form of mRNA is in vitro translated and stable ribosomal complexes linking the phenotype (folded DARPins) with the genotype (translated mRNA) are generated. The ribosomal complexes are allowed to bind to immobilized bLmrCDAviC. After a washing step of variable length (depending on selection stringency), bound ribosomal complexes are destabilized and mRNA encoding for potential target-specific DARPins is liberated. The eluted mRNA is amplified by reverse transcription and PCR to double stranded DNA, which is in vitro transcribed into mRNA for another round of selection or used for binder analysis. (B) Schematic drawing of the ELISA set up. Protein A is coated onto the ELISA well and is decorated with an anti-myc antibody that immobilizes the DARPins via the C-terminal Myc5-tag. Upon binding of purified, biotinylated target protein (e.g. LmrCD, AcrB or MsbA in our study) to DARPin, the target protein is detected using a streptavidin-alkaline phosphatase the activity of which was detected colourimetrically at OD405 using p-nitrophenyl phosphate as a substrate.
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pone-0037845-g001: Ribosome display and ELISA set-up.(A) Sketch delineating one DARPin selection round using ribosome display (adopted from [31]). The DARPin library in form of mRNA is in vitro translated and stable ribosomal complexes linking the phenotype (folded DARPins) with the genotype (translated mRNA) are generated. The ribosomal complexes are allowed to bind to immobilized bLmrCDAviC. After a washing step of variable length (depending on selection stringency), bound ribosomal complexes are destabilized and mRNA encoding for potential target-specific DARPins is liberated. The eluted mRNA is amplified by reverse transcription and PCR to double stranded DNA, which is in vitro transcribed into mRNA for another round of selection or used for binder analysis. (B) Schematic drawing of the ELISA set up. Protein A is coated onto the ELISA well and is decorated with an anti-myc antibody that immobilizes the DARPins via the C-terminal Myc5-tag. Upon binding of purified, biotinylated target protein (e.g. LmrCD, AcrB or MsbA in our study) to DARPin, the target protein is detected using a streptavidin-alkaline phosphatase the activity of which was detected colourimetrically at OD405 using p-nitrophenyl phosphate as a substrate.

Mentions: We cloned the lmrCD genes with a His10-tag N-terminally to LmrC, and were able to purify functionally active LmrCD to homogeneity from lactococcal membrane vesicles. The proteins could be isolated as heterodimeric species from size exclusion chromatography (SEC) columns (Figure S1A and B). Interestingly, the heterodimeric complex of LmrCD was stable when the purified protein was analyzed by nano-electrospray mass spectrometry [50]. In order to immobilize LmrCD during the DARPin selection procedure, an Avi-tag was introduced C-terminally to LmrD, which allowed for site-specific enzymatic biotinylation of a lysine residue comprised within the Avi-tag sequence (biotinylated LmrCD is denoted bLmrCDAviC) [51]. The DARPin selection was performed using the ribosome display method with DARPins including three internal randomized repeats (N3C DARPins) (Figure 1A) [12], [18], [28]. A total of 4 sequential selection rounds were performed in which catalytically active bLmrCDAviC and orthovanadate-trapped bLmrCDAviC were used as two independent protein formulations. In the presence of 1 mM ATP, LmrCD could be trapped by orthovanadate with a concentration giving half-maximal inhibition of ATP hydrolysis (IC50) of 120 µM which is in agreement with a recent study on the heterodimeric ABC transporter BmrCD [52] (data not shown). The orthovanadate concentration (1 mM) used during the DARPin selections comfortably exceeded this IC50. It should be noted that around 0.6 mM of ATP originating from the in vitro translation buffer and around 40 mM magnesium acetate were present during the incubation of the DARPins with the target protein. This means that in case of the non-trapped bLmrCDAviC formulation, the DARPins were selected against transporters slowly hydrolyzing ATP and presumably adopting various conformational states.


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)

Ribosome display and ELISA set-up.(A) Sketch delineating one DARPin selection round using ribosome display (adopted from [31]). The DARPin library in form of mRNA is in vitro translated and stable ribosomal complexes linking the phenotype (folded DARPins) with the genotype (translated mRNA) are generated. The ribosomal complexes are allowed to bind to immobilized bLmrCDAviC. After a washing step of variable length (depending on selection stringency), bound ribosomal complexes are destabilized and mRNA encoding for potential target-specific DARPins is liberated. The eluted mRNA is amplified by reverse transcription and PCR to double stranded DNA, which is in vitro transcribed into mRNA for another round of selection or used for binder analysis. (B) Schematic drawing of the ELISA set up. Protein A is coated onto the ELISA well and is decorated with an anti-myc antibody that immobilizes the DARPins via the C-terminal Myc5-tag. Upon binding of purified, biotinylated target protein (e.g. LmrCD, AcrB or MsbA in our study) to DARPin, the target protein is detected using a streptavidin-alkaline phosphatase the activity of which was detected colourimetrically at OD405 using p-nitrophenyl phosphate as a substrate.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0037845-g001: Ribosome display and ELISA set-up.(A) Sketch delineating one DARPin selection round using ribosome display (adopted from [31]). The DARPin library in form of mRNA is in vitro translated and stable ribosomal complexes linking the phenotype (folded DARPins) with the genotype (translated mRNA) are generated. The ribosomal complexes are allowed to bind to immobilized bLmrCDAviC. After a washing step of variable length (depending on selection stringency), bound ribosomal complexes are destabilized and mRNA encoding for potential target-specific DARPins is liberated. The eluted mRNA is amplified by reverse transcription and PCR to double stranded DNA, which is in vitro transcribed into mRNA for another round of selection or used for binder analysis. (B) Schematic drawing of the ELISA set up. Protein A is coated onto the ELISA well and is decorated with an anti-myc antibody that immobilizes the DARPins via the C-terminal Myc5-tag. Upon binding of purified, biotinylated target protein (e.g. LmrCD, AcrB or MsbA in our study) to DARPin, the target protein is detected using a streptavidin-alkaline phosphatase the activity of which was detected colourimetrically at OD405 using p-nitrophenyl phosphate as a substrate.
Mentions: We cloned the lmrCD genes with a His10-tag N-terminally to LmrC, and were able to purify functionally active LmrCD to homogeneity from lactococcal membrane vesicles. The proteins could be isolated as heterodimeric species from size exclusion chromatography (SEC) columns (Figure S1A and B). Interestingly, the heterodimeric complex of LmrCD was stable when the purified protein was analyzed by nano-electrospray mass spectrometry [50]. In order to immobilize LmrCD during the DARPin selection procedure, an Avi-tag was introduced C-terminally to LmrD, which allowed for site-specific enzymatic biotinylation of a lysine residue comprised within the Avi-tag sequence (biotinylated LmrCD is denoted bLmrCDAviC) [51]. The DARPin selection was performed using the ribosome display method with DARPins including three internal randomized repeats (N3C DARPins) (Figure 1A) [12], [18], [28]. A total of 4 sequential selection rounds were performed in which catalytically active bLmrCDAviC and orthovanadate-trapped bLmrCDAviC were used as two independent protein formulations. In the presence of 1 mM ATP, LmrCD could be trapped by orthovanadate with a concentration giving half-maximal inhibition of ATP hydrolysis (IC50) of 120 µM which is in agreement with a recent study on the heterodimeric ABC transporter BmrCD [52] (data not shown). The orthovanadate concentration (1 mM) used during the DARPin selections comfortably exceeded this IC50. It should be noted that around 0.6 mM of ATP originating from the in vitro translation buffer and around 40 mM magnesium acetate were present during the incubation of the DARPins with the target protein. This means that in case of the non-trapped bLmrCDAviC formulation, the DARPins were selected against transporters slowly hydrolyzing ATP and presumably adopting various conformational states.

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