The transport mechanism of the multidrug transporter AcrB involves two binding areas that are seperated by a switch-loop

AcrAB-TolC is the major efflux protein complex in Escherichia coli extruding a vast variety of antimicrobial agents from the cell. The functional unit of inner membrane component AcrB is a homotrimer and the monomers adopt three different conformations designated loose (L), tight (T) and open (O) ¹. It is postulated that the AcrB monomers cycle consecutively through these three conformational stages in a concerted fashion, from L to T to O and back to L, much in analogy to the functional rotation observed for the synthesis of ATP by the F1FO-ATPase ². For the asymmetric AcrB homotrimer, binding of drugs has been shown in the T monomer at an internal hydrophobic binding pocket ³. The initial drug binding step and the transport towards the internal periplasmic drug binding site in the T conformation presumably mediated by an intrinsic tunnel system has been elusive thus far due to the lack of high resolution AcrB/drug co-crystal structures. We report high resolution structures (2.0-2.25 Å) of AcrB/DARPin complexes with bound minocyclin or doxorubicin. The results indicate a stepwise drug transport with initial binding to the L monomer and further transport mediated by a flexible switch-loop to the internal deep binding pocket of the T monomer. Proton-motive force driven conformational change to the O state resulting in the release of the bound drug towards the outside of the cell and the return to the L state concludes the functional rotation cycle.