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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.

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Identification and characterization of DARPin binders by ELISA(A) Specificity ELISA using bLmrCDAviC, bMsbAAviC and bAcrBAviC as target proteins. Seven DARPins (α-LmrCD#1-5, DARPin_Act2 and DARPin_Act3) were found to be highly specific for bLmrCDAviC. Many initial DARPin binder-hits promiscuously bound to bLmrCDAviC, bMsbAAviC and bAcrBAviC as exemplified with the “unsp. DARPin” and were therefore not useful for further analysis. DARPins specific for bMsbAAviC (DARPin_55) and bAcrBAviC (110819) were used as a positive control. (B) ELISA analyzing binding of the LmrCD-specific DARPins shown in (A) to LmrC (bLmrC-GFP), LmrD (bLmrD-GFP) and the nucleotide binding domain of LmrD (bLmrD-NBDAviN). Binding to LmrCD (bLmrCDAviC) was confirmed as positive control.
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pone-0037845-g003: Identification and characterization of DARPin binders by ELISA(A) Specificity ELISA using bLmrCDAviC, bMsbAAviC and bAcrBAviC as target proteins. Seven DARPins (α-LmrCD#1-5, DARPin_Act2 and DARPin_Act3) were found to be highly specific for bLmrCDAviC. Many initial DARPin binder-hits promiscuously bound to bLmrCDAviC, bMsbAAviC and bAcrBAviC as exemplified with the “unsp. DARPin” and were therefore not useful for further analysis. DARPins specific for bMsbAAviC (DARPin_55) and bAcrBAviC (110819) were used as a positive control. (B) ELISA analyzing binding of the LmrCD-specific DARPins shown in (A) to LmrC (bLmrC-GFP), LmrD (bLmrD-GFP) and the nucleotide binding domain of LmrD (bLmrD-NBDAviN). Binding to LmrCD (bLmrCDAviC) was confirmed as positive control.

Mentions: We analyzed 190 clones from the DARPin pools, enriched over four selection rounds against untreated or vanadate-trapped bLmrCDAviC, by an established ELISA protocol (95 DARPins for each protein formulation) (Figure 1B, Figure 2) [31]. From the initial ELISA (not shown) we chose the clones giving rise to the 30 most intense ELISA signals against bLmrCDAviC (15.8% of examined clones) for a second comparative ELISA (Figure 3A). Besides LmrCD, the ABC transporter MsbA and the secondary-active multidrug transporter AcrB were used in the assay (prepared as proteins biotinylated at the C-terminal Avi-tag). From the 30 ELISA-positive DARPins, 8 were exclusively binding to bLmrCDAviC but not to bMsbAAviC or bAcrBAviC (4.2% of all examined clones), whereas the other 22 DARPins were promiscuously binding to all membrane proteins used in the specificity ELISA (Figure 3A). The quality of the control proteins bMsbAAviC and bAcrBAviC was confirmed by using target-specific DARPins in the ELISA assay (AcrB-specific DARPin 110819 is described [18]; the selection of the MsbA-specific DARPin_55 will be published elsewhere). The genes encoding the eight LmrCD-specific DARPins were sub-cloned, expressed without the C-terminal Myc5-tag and analyzed by size exclusion chromatography. Four of these DARPins displayed a substantial degree of aggregation (soluble aggregates) and were therefore excluded. The other four LmrCD-specific DARPins (α-LmrCD#1-4) ran as monomeric or dimeric species on SEC taking the elution profile of the monomeric control DARPin E3_5 as a reference (Table 1, Figure S1C). Three out of these four DARPins exhibited tight binding to purified LmrCD, and eluted in complex with their target from the size exclusion column. Thus, the initially chosen 190 DARPin clones could be narrowed down to 3 specific high-affinity binders, corresponding to a hit rate of 1.6%. A fifth high-affinity DARPin (α-LmrCD#5) was found in another ELISA screen identical to the one above (not shown).


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)

Identification and characterization of DARPin binders by ELISA(A) Specificity ELISA using bLmrCDAviC, bMsbAAviC and bAcrBAviC as target proteins. Seven DARPins (α-LmrCD#1-5, DARPin_Act2 and DARPin_Act3) were found to be highly specific for bLmrCDAviC. Many initial DARPin binder-hits promiscuously bound to bLmrCDAviC, bMsbAAviC and bAcrBAviC as exemplified with the “unsp. DARPin” and were therefore not useful for further analysis. DARPins specific for bMsbAAviC (DARPin_55) and bAcrBAviC (110819) were used as a positive control. (B) ELISA analyzing binding of the LmrCD-specific DARPins shown in (A) to LmrC (bLmrC-GFP), LmrD (bLmrD-GFP) and the nucleotide binding domain of LmrD (bLmrD-NBDAviN). Binding to LmrCD (bLmrCDAviC) was confirmed as positive control.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3366976&req=5

pone-0037845-g003: Identification and characterization of DARPin binders by ELISA(A) Specificity ELISA using bLmrCDAviC, bMsbAAviC and bAcrBAviC as target proteins. Seven DARPins (α-LmrCD#1-5, DARPin_Act2 and DARPin_Act3) were found to be highly specific for bLmrCDAviC. Many initial DARPin binder-hits promiscuously bound to bLmrCDAviC, bMsbAAviC and bAcrBAviC as exemplified with the “unsp. DARPin” and were therefore not useful for further analysis. DARPins specific for bMsbAAviC (DARPin_55) and bAcrBAviC (110819) were used as a positive control. (B) ELISA analyzing binding of the LmrCD-specific DARPins shown in (A) to LmrC (bLmrC-GFP), LmrD (bLmrD-GFP) and the nucleotide binding domain of LmrD (bLmrD-NBDAviN). Binding to LmrCD (bLmrCDAviC) was confirmed as positive control.
Mentions: We analyzed 190 clones from the DARPin pools, enriched over four selection rounds against untreated or vanadate-trapped bLmrCDAviC, by an established ELISA protocol (95 DARPins for each protein formulation) (Figure 1B, Figure 2) [31]. From the initial ELISA (not shown) we chose the clones giving rise to the 30 most intense ELISA signals against bLmrCDAviC (15.8% of examined clones) for a second comparative ELISA (Figure 3A). Besides LmrCD, the ABC transporter MsbA and the secondary-active multidrug transporter AcrB were used in the assay (prepared as proteins biotinylated at the C-terminal Avi-tag). From the 30 ELISA-positive DARPins, 8 were exclusively binding to bLmrCDAviC but not to bMsbAAviC or bAcrBAviC (4.2% of all examined clones), whereas the other 22 DARPins were promiscuously binding to all membrane proteins used in the specificity ELISA (Figure 3A). The quality of the control proteins bMsbAAviC and bAcrBAviC was confirmed by using target-specific DARPins in the ELISA assay (AcrB-specific DARPin 110819 is described [18]; the selection of the MsbA-specific DARPin_55 will be published elsewhere). The genes encoding the eight LmrCD-specific DARPins were sub-cloned, expressed without the C-terminal Myc5-tag and analyzed by size exclusion chromatography. Four of these DARPins displayed a substantial degree of aggregation (soluble aggregates) and were therefore excluded. The other four LmrCD-specific DARPins (α-LmrCD#1-4) ran as monomeric or dimeric species on SEC taking the elution profile of the monomeric control DARPin E3_5 as a reference (Table 1, Figure S1C). Three out of these four DARPins exhibited tight binding to purified LmrCD, and eluted in complex with their target from the size exclusion column. Thus, the initially chosen 190 DARPin clones could be narrowed down to 3 specific high-affinity binders, corresponding to a hit rate of 1.6%. A fifth high-affinity DARPin (α-LmrCD#5) was found in another ELISA screen identical to the one above (not shown).

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