Cooperativity and allostery within catalytically active protein complexes are important concepts for control of phenotypic outcome within a living system. In this chapter, the putative molecular determinants for cooperativity and allostery of a multi-subunit antibiotic efflux pump complex are discussed. While resisting multiple antibiotic stresses, Gram-negative bacteria deploy a network of single and multicomponent drug efflux pumps to reduce the concentration of the drugs in the cytoplasm and periplasm. The dual membrane setup imposes a particular challenge for transport of drugs from the cytoplasm to the medium, and current hypothesis describes a multistep transport path of drugs across the inner and outer membrane by the action of different drug efflux pumps. Drug transport from the cytoplasm to the periplasm is catalyzed by single component drug efflux systems belonging to the ABC-transporter superfamily, the Major Facilitator Superfamily (MFS), The Multi Antimicrobial Extrusion (MATE) Family, and the Small Multidrug Resistance (SMR) family. Transport from the periplasm across the outer membrane is catalyzed by a tripartite transport complex consisting of an inner membrane Resistance-Nodulation-cell Division (RND) transporter, an adaptor protein, and an outer membrane channel. Cooperative effects occur at multiple levels within this three component RND system: Anticipated allostery for binding of ligands (drugs and H+) at the level of the protomer, interdependence of the protomers within the trimer, and cooperative effects between the three components within the entire transport complex. The molecular determinants of multiple substrates binding at different sites within the inner membrane transporter and its coupling to H+ binding and transport are being described here for the paradigm tripartite transport machinery AcrAB-TolC from Escherichia coli. High resolution structures of the three components, the anticipated three component setup, and a multitude of biophysical and biochemical data are combined to address the overall molecular understanding of secondary H+/drug antiport. With focus on the inner membrane RND component, drug and H+ binding and transport cooperativity are exemplified by exploiting a mechanism based on binding change and the strict coupling to the influx of H+.
Multidrug efflux Functional rotation Cooperativity Tripartite RND systems Proton translocation Drug/H+ antiporter Multiple drug binding Conformational changes Transporter states Membrane transport
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The work of the Pos lab presented in this chapter was supported by the Swiss National Foundation, the German Research Foundation (SFB 807, Transport and Communication across Biological Membranes), the DFG-EXC115 (Cluster of Excellence Macromolecular Complexes at the Goethe-University Frankfurt), the Innovative Medicine Initiative (IMI), Project TRANSLOCATION (http://www.imi.europa.eu/content/translocation) and by grants from Europe Aspire and Human Frontier Science Program.
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