The acquisition and maintenance of antibiotic resistance in Gram-negative bacteria is heavily dependent on the presence of so called Resistance Nodulation cell Division (RND) superfamily transporter complexes. These tripartite protein assemblies mediate the ion-motive force driven efflux of diverse toxic compounds, such as bile salts, detergents, organic solvents, and many structurally unrelated antibiotics. Drug transport by the RND system archetype, the AcrA/AcrB/TolC complex from Escherichia coli, has shown to be highly cooperative. In the framework of the CRC, we will investigate the structural determinants for assembly, ion specificity, and transport cooperativity for the drug/H+ antiporter AcrA/AcrB/TolC complex from E. coli and the drug/Na+ antiporter VexE/VexF/TolC complex from Vibrio cholerae. Structural analysis via X-ray crystallography will be continuously employed as an accompanying tool to visualize the molecular basis of these three areas under investigation. The inner membrane components AcrB and VexF form homotrimeric assemblies which are key to coupling-ion specificity, allosteric drug binding and cooperative drug transport. The transmembrane domains of the AcrB and VexF determine the coupling-ion specificity of the entire tripartite system. By means of constructing hybrid AcrB/VexF transporters, the major aim will be to gradually identify those amino acid side chain residues responsible for Na+ or H+ coupling. In combination, cooperative features of these transport systems will be determined by drug and ion transport measurements in the whole bacterial cell systems and in the reconstituted proteoliposomal system.
Fig. 2: The access and deep binding pockets with bound doxorubicin in the asymmetric AcrB trimer. The protomers are colored in blue (L monomer), yellow (T) or red (O). Putative substrate entrance/exit tunnels are visualized in green. A) Side view of the asymmetric AcrB trimer with two doxorubicin molecules (yellow spheres) bound to the access pocket in the L monomer and in the same trimer one doxorubicin (blue spheres) bound at the deep binding pocket in the T monomer. B) Periplasmic top-view as shown in A) (Eicher et al 2012 PNAS).
Du D, van Veen HW, Murakami S, Pos KM, Luisi B (2015) Structure, mechanism and cooperation of bacterial multidrug transporters. Curr Opin Struc Biol 33, 76-91.
Müller RT, Pos KM (2015) The assembly and disassembly of the AcrAB-TolC three-component multidrug efflux pump. Biol Chem 396, 1083-89.
Ly K, Bartho JD, Eicher T, Pos KM, Mitra AK (2014) A novel packing arrangement of AcrB in the lipid bilayer membrane. FEBS Lett 588, 4776-83.
Eicher T, Seeger MA, Anselmi C, Zhou W, Brandstätter L, Verrey F, Diederichs K, Faraldo-Gómez JD*, Pos KM* (2014) Coupling of remote alternating-access transport mechanisms for protons and substrates in the multidrug efflux pump AcrB. eLife 3, e03145.
Cha H, Pos KM (2014) Cooperative transport mechanism and proton-coupling in the multidrug efflux transporter complex AcrAB-TolC. Membrane Transport Mechanism – 3D structure and beyond. Ed. Krämer R. and Ziegler C. Springer Series in Biophysics 17, 207-32.
Cha H, Müller RT, Pos KM (2014) Switch-loop flexibility affects transport of large drugs by the promiscuous AcrB multidrug efflux transporter. Antimicrob Agents Chemother 58, 4767-72.
Ruggerone P, Murakami S, Pos KM, Vargiu AV (2013) RND Efflux Pumps: Structural information translated into function and inhibition mechanisms. Curr Top Med Chem 13, 3079-3100.
Ong YS, Lakatos A, Becker-Baldus J, Pos KM, Glaubitz C (2013) Detecting substrates bound to the secondary multidrug efflux pump EmrE by DNP enhanced solid-state NMR. J Am Chem Soc 135, 15754-62.
Hahn A, Stevanovic M, Mirus O, Lytvynenko I, Pos KM, Schleiff E (2013) The outer Membrane TolC-like channel HgdD is part of tripartite Resistance Nodulation cell Division (RND) efflux systems conferring multiple drug resistance in the cyanobacterium Anabaena sp PCC7120. J Biol Chem 288, 31192-205.
Du D, Venter H, Pos KM, Luisi BF (2013) The Machinery and Mechanism of Multidrug Efflux in Gram-negative Bacteria. Microbial Efflux Pumps: Current Research. Chapter 3.
Eicher T, Cha H, Seeger MA, Brandstätter L, Bohnert JA, Kern WV, Verrey F, Grütter MG, Diederichs K, Pos KM (2012) Transport of drugs by the multidrug transporter AcrB involves an access and a deep binding pocket that are separated by a switch-loop. Proc Nat Acad Sci USA 109, 5687-5692.