Drug Export Pathway of Multidrug Exporter AcrB Revealed by DARPin Inhibitors

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Abstract
The multidrug exporter AcrB is the inner membrane component of the AcrAB-TolC drug efflux system in Escherichia coli and is responsible for the resistance of this organism to a wide range of drugs. Here we describe the crystal structure of the trimeric AcrB in complex with a designed ankyrin-repeat protein (DARPin) inhibitor at 2.5-Å resolution. The three subunits of AcrB are locked in different conformations revealing distinct channels in each subunit. There seems to be remote conformational coupling between the channel access, exit, and the putative proton-translocation site, explaining how the proton motive force is used for drug export. Thus our structure suggests a transport pathway not through the central pore but through the identified channels in the individual subunits, which greatly advances our understanding of the multidrug export mechanism. Bacterial resistance to antibiotics is a major challenge for the current treatment of infectious diseases. One way bacteria can escape destruction is by pumping out administered drugs through specific transporter proteins that span the cell membrane. We used designer proteins that bind to and stabilize proteins of interest in order to study the major drug efflux pump of Escherichia coli, AcrB. After selecting for designed ankyrin repeat proteins (DARPins) that inhibit this pump, we determined the crystal structure of a DARPin inhibitor in complex with AcrB. We confirmed that the AcrB is split into three subunits, each of which exhibits distinctly different conformations. Moreover, we show that each subunit has a differently shaped substrate transport channel; these variable channels provide unique snapshots of the different conformations adopted by AcrB during transport of a substrate. The structure also offers an explanation for how substrate export is structurally coupled to simultaneous proton import—thus significantly improving our understanding of the mechanism of AcrB. This is the first report of the selection and co-crystallization of a DARPin with a membrane protein, which demonstrates the potential of DARPins not only as inhibitors but also as tools for the structural investigation of integral membrane proteins.