Tuning the drug efflux activity of an ABC transporter in vivo by in vitro selected DARPin binders.
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.
Department of Pharmacology, University of Cambridge, Cambridge, United Kingdom. m.seeger@bioc.uzh.ch
Markus A
MA
Seeger
Anshumali
A
Mittal
Saroj
S
Velamakanni
Michael
M
Hohl
Stefan
S
Schauer
Ihsene
I
Salaa
Markus G
MG
Grütter
Hendrik W
HW
van Veen
D018528:ATP-Binding Cassette Transporters / Q000378:metabolism*; D000251:Adenosine Triphosphatases / Q000378:metabolism; D017089:Ankyrin Repeat; D001692:Biological Transport / Q000187:drug effects; D055592:Biophysical Phenomena / Q000187:drug effects; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D003630:Daunorubicin / Q000494:pharmacology; D003902:Detergents / Q000494:pharmacology; D004797:Enzyme-Linked Immunosorbent Assay; D018604:Epitope Mapping; D000939:Epitopes / Q000276:immunology; D013294:Lactococcus lactis / Q000187:drug effects / Q000378:metabolism*; D004364:Pharmaceutical Preparations / Q000378:metabolism*; D011485:Protein Binding / Q000187:drug effects; D011510:Proteolipids / Q000187:drug effects / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism*; D012270:Ribosomes / Q000187:drug effects / Q000378:metabolism; D012995:Solubility / Q000187:drug effects; D020349:Surface Plasmon Resonance
D018528:ATP-Binding Cassette Transporters; D003902:Detergents; D000939:Epitopes; D004364:Pharmaceutical Preparations; D011510:Proteolipids; D011994:Recombinant Proteins; C020485:proteoliposomes; D000251:Adenosine Triphosphatases; D003630:Daunorubicin