Tuning the drug efflux activity of an ABC transporter in vivo by in vitro selected DARPin binders.
Author(s)
Type
Journal Article
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.
Date Issued
2012-06-04
Date Acceptance
2012-04-30
Start Page
e37845
Journal / Book Title
PLoS One
Volume
7
Issue
6
Copyright Statement
© 2012 Seeger et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Identifier
https://www.ncbi.nlm.nih.gov/pubmed/22675494
PONE-D-12-02082
Subjects
ATP-Binding Cassette Transporters
Adenosine Triphosphatases
Ankyrin Repeat
Biological Transport
Biophysical Phenomena
Cell Membrane
Daunorubicin
Detergents
Enzyme-Linked Immunosorbent Assay
Epitope Mapping
Epitopes
Lactococcus lactis
Pharmaceutical Preparations
Protein Binding
Proteolipids
Recombinant Proteins
Ribosomes
Solubility
Surface Plasmon Resonance
MD Multidisciplinary
General Science & Technology
Publication Status
Published
Coverage Spatial
United States