Asymmetry in the homodimeric ABC transporter MsbA recognized by a DARPin

J Biol Chem. 2012 Jun 8;287(24):20395-406. doi: 10.1074/jbc.M112.359794. Epub 2012 Apr 20.


ABC transporters harness the energy from ATP binding and hydrolysis to translocate substrates across the membrane. Binding of two ATP molecules at the nucleotide binding domains (NBDs) leads to the formation of an outward-facing state. The conformational changes required to reset the transporter to the inward-facing state are initiated by sequential hydrolysis of the bound nucleotides. In a homodimeric ABC exporter such as MsbA responsible for lipid A transport in Escherichia coli, sequential ATP hydrolysis implies the existence of an asymmetric conformation. Here we report the in vitro selection of a designed ankyrin repeat protein (DARPin) specifically binding to detergent-solubilized MsbA. Only one DARPin binds to the homodimeric transporter in the absence as well as in the presence of nucleotides, suggesting that it recognizes asymmetries in MsbA. DARPin binding increases the rate of ATP hydrolysis by a factor of two independent of the substrate-induced ATPase stimulation. Electron paramagnetic resonance (EPR) measurements are found to be in good agreement with the available crystal structures and reveal that DARPin binding does not affect the large nucleotide-driven conformational changes of MsbA. The binding epitope was mapped by cross-linking and EPR to the membrane-spanning part of the transmembrane domain (TMD). Using cross-linked DARPin-MsbA complexes, 8-azido-ATP was found to preferentially photolabel one chain of the homodimer, suggesting that the asymmetries captured by DARPin binding at the TMDs are propagated to the NBDs. This work demonstrates that in vitro selected binders are useful tools to study the mechanism of membrane proteins.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • ATP-Binding Cassette Transporters / chemistry*
  • ATP-Binding Cassette Transporters / genetics
  • ATP-Binding Cassette Transporters / metabolism
  • Adenosine Triphosphate / chemistry
  • Adenosine Triphosphate / genetics
  • Adenosine Triphosphate / metabolism
  • Bacterial Proteins / chemistry*
  • Bacterial Proteins / genetics
  • Bacterial Proteins / metabolism
  • Biological Transport, Active
  • Electron Spin Resonance Spectroscopy
  • Escherichia coli / chemistry*
  • Escherichia coli / genetics
  • Escherichia coli / metabolism
  • Lipid A / chemistry
  • Lipid A / genetics
  • Lipid A / metabolism
  • Protein Binding
  • Protein Multimerization*
  • Protein Structure, Tertiary


  • ATP-Binding Cassette Transporters
  • Bacterial Proteins
  • Lipid A
  • MsbA protein, Bacteria
  • Adenosine Triphosphate