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)-type superfamily 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. The RND systems comprise a promiscuous drug binding and transporting inner membrane protein, energized by the electrochemical gradient of ions across the membrane, a periplasmic adaptor protein, and an outer membrane channel.
In the framework of the CRC 807, we will investigate the structural determinants for the promiscuous substrate binding, molecular setup, and transport activity of tripartite RND complexes from selected Gram-negative bacteria. Via X-ray crystallography and other biophysical and biochemical methods, we obtained molecular insights into the promiscuous binding phenotype of various drug substrates and inhibitors with the E. coli inner membrane component AcrB. Despite that RND complex assembly and disassembly is a highly dynamic process, we have obtained a single-particle EM model of the entire tripartite AcrAB-TolC reconstituted in nanodiscs.
In the upcoming funding period, we aim to examine the molecular basis of multiple drug-binding phenotypes of homologue RND components and the molecular setup of tripartite assemblies from other Gram-negative bacteria by using (high-resolution) single-particle cryo-EM (in collaboration with Kühlbrandt, P01), solid-state NMR (in collaboration with Glaubitz, P06), EPR (in collaboration with Prisner/Joseph, P07), and X-ray crystallography. The in vitro formation of tripartite assemblies will allow us to characterize drug transport in a reconstituted system and AcrB variants with altered drug binding specificity will enable the detailed analysis on the molecular basis of promiscuous drug binding. The combined results from the proposed research will provide detailed insight into the structure-function relationship of RND-type tripartite multidrug efflux complexes.
High-resolution structures of the AcrB periplasmic domain in complex with Rhodamine 6G (2.65 Å) and the pyranopyridine inhibitor (1.8 Å).
(a) Side-view on the asymmetric (LLT) AcrBper. Protomers in Loose conformation (L) shown in blue, protomer in Tight conformation (T) shown in yellow. Close-up view of (b) Rhodamine 6G and (c) the pyranopyridine inhibitor bound in the deep binding pocket and hydrophobic trap. The 2Fo-Fc densities of the bound compounds are indicated in blue mesh.
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.