Membrane Transport Machineries: The Antibiotic Efflux Pump AcrA/AcrB/TolC
Bacteria have a plethora of mechanisms to protect themselves against noxious compounds like antibiotics. Many of them possess drug efflux pumps to extrude drugs out of the cell and keep the therapeutic concentration below the toxic threshold so the bacteria will survive. These drug efflux systems play key roles in many multi-resistant pathogens causing serious problems especially for those of us which are immunocompromised (through e.g. chemotherapy or HIV-infection).
The research group "Membrane Transport Machineries" at the Institute of Biochemistry focuses on the structural elucidation of these pumps, in order to obtain molecular information on their transport mechanisms. These consolidated findings might be useful for the development of antibiotic efflux inhibiting compounds.
The AcrA/AcrB/TolC antibiotic efflux pump from Escherichia coli is a tripartite system which spans not only the inner membrane, but also the periplasm and outer membrane of this Gram-negative bacterium (Figure 1).
Figure 1: Schematic Drawing of the AcrAB-TolC antibiotic efflux pump of Escherichia coli. The inner membrane component AcrB (blue) is both the substrate specificity determinant and the energy module of the AcrA/AcrB/TolC efflux system. Drugs are transported from the outer leaflet of the inner membrane via a coupled drug/proton antiport mechanism. TolC (yellow) forms a channel in the outer membrane. AcrA (red) is an adaptor connecting AcrB and TolC.
This three component system is comprised of an inner membrane located substrate/proton antiporter, designated "Acriflavine resistance protein B" (AcrB), a channel located in the outer membrane "Tolerance Colicin E1" (TolC), as well as an adapter protein located in the periplasm called "Acriflavine resistance protein A" (AcrA). Dysfunction of any of the components leads to a drastic reduction of the natural resistance of the bacterium towards antibiotics, bile salts, detergents and dyes.
AcrB plays a central role for both substrate specificity and energy transduction. It is a member of the Resistance Nodulation and cell Division (RND) Superfamily, which includes the human Niemann-Pick C1 (NPC1) and Hedgehog Receptor Patched (Ptc).
We were able in a collaborative effort with Prof. Kay Diederichs (Konstanz) to elucidate a high resolution structure of AcrB in an asymmetric conformation.
Based on the asymmetry, a new transport mechanism could be postulated for the transport of drugs through the single monomers. The new structure reveals tunnels inside the monomers which contain gorge portions (Figure 2). A model for the transport of antibiotics mediated by AcrB over the bacterial membrane reminiscent to the mechanism of a peristaltic pump is postulated.
Figure 2: Visualization of tunnels in the porter (pore) domain of the trimeric AcrB peristaltic drug efflux pump. The AcrB monomers are presented in (a) blue (loose), (b) yellow (tight) and (c) red (open). The tunnels are highlighted as green surfaces in a ribbon model of the AcrB trimer. Inset: Bound minocyclin is depicted inside a hydrophobic pocket in the tight monomer (yellow) with the observed electron density in a 2Fo-Fc electron density map contoured 1 σ The Panels on the left and right represent in each case a one third conversion of a full loose→tight→open→loose cycle.
Since AcrB is driven by the proton motive force, the energy conversion to drive the large conformational changes observed in the periplasmic domain is considered to be generated by protonation and deprotonation of essential charged residues residing in the transmembrane domain. Our research is now focused on the putative pathway for protons through the transmembrane domain and its coupling to the events during functional rotation leading to drug export.
For more information: