Principal Investigator
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P18 Klaas Martinus Pos
Professor
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Institute of Biochemistry
Biocenter
Goethe-University Frankfurt a.M.
Max-von-Laue-Str. 9
60438 Frankfurt am Main, Germany
Phone +49 (0)69 79 82 92 51
Fax +49 (0)69 79 82 92 01
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P18 Structure and function of Resistance Nodulation and cell Division (RND) transporters and RND-type tripartite complexes
The AcrA/AcrB/TolC tripartite protein complex is the major drug resistance pump of Escherichia coli and mediates the proton-motive force driven efflux of diverse substrates, such as bile salts, detergents, organic solvents and many structurally unrelated antibiotics. The inner membrane component AcrB is a member of the Resistance Nodulation cell Division (RND) superfamily, which includes the human homologues Niemann Pick transporter type C1 and the hedgehog receptor Patched. Within the three component efflux system, AcrB is central to substrate selectivity and energy transduction and is the current paradigm for RND superfamily structure and function. The most recent X-ray structures of trimeric AcrB revealed three different conformations of the monomers representing consecutive steps of a transport cycle involving the creation of alternate access tunnels to and from a hydrophobic substrate binding pocket.
In the framework of the SFB, we will investigate the structural determinants for substrate selectivity, ion coupling and the coupling between the conformational changes in the membrane part of AcrB (due to proton influx) to those observed in the periplasmic part, which lead to antibiotic efflux. We are extending this structural and functional studies to the complete pump complex of AcrAB-TolC and homologue tripartite systems from proteobacterial sources.
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Fig. 1: Schematic drawing of the tri-partite setup of efflux pumps in Gram-negative bacteria exemplified by the AcrAB-TolC efflux pump from E. coli.
Fig. 2: Tunnels in the porter domain of trimeric AcrB peristaltic drug efflux pump. The AcrB monomers are presented in blue (loose), yellow (tight) and red (open). The tunnels are highlighted as green surfaces in a ribbon model of the AcrB trimer and might function as transport paths of drugs. Tunnel 1 might serve as entrance for drugs from the outer leaflet of the inner membrane towards the hydrophobic substrate binding pocket. Tunnel 2 might serve as an alternative entrance for substrates entering via the periplasm or as exit duct for nonsubstrates. Tunnel 3 in the open monomer is the exit pathway for substrates towards TolC and the outside medium. Inset: In the T monomer (yellow), a hydrophobic pocket is defined by phenylalanine, valine, isoleucine and tyrosine side chains at the PN2/PC1 interface. Bound minocyclin is depicted with the observed electron density in a 2Fo-Fc electron density map contoured 1 σ (Eicher, Seeger, Pos et al, unpublished). Panels A and B represent in each case a one third conversion of a full L→T→O→L cycle. (Adapted from Pos (2009) and modified)
Fig. 3: Structure of AcrB. (A) Side view of symmetric AcrB trimer illustrated by a backbone ribbon diagram. From top to bottom: the TolC docking domain includes a funnel-like structure. It narrows to a central pore located in the pore domain. The transmembrane domain confines a central cavity. (B) Top view from the periplasm along the crystallographic three-fold axis. The long loops protruding from one monomer into the next provide the main interaction within the AcrB trimer. (C) View as in (B) but the TolC docking domain has been removed. Subdomains PN1, PN2, PC1 and PC2 are indicated. The lateral cleft between subdomains PC1 and PC2 is suggested to accommodate AcrA. (D) Topology of the transmembrane helices top view along the crystallographic three-fold axis. The three monomers enclose a 30-35 Å wide central cavity. The TM4 and TM10 helices are surrounded by the other TM helices in each monomer and contain the titratable residues Asp407, Asp408 (TM4) and Lys940 (TM10). The structurally identical protomers (A-D) are individually coloured.
Fig. 4: Main Structural differences of the AcrB monomers. A) Side view of the proton relay. In the loose and tight state (blue), D408 and D407 sandwich K940 and R971 is oriented towards the triad. The polar side chains of D407, D408 K940 and T978 are forming a continuous hydrogen bonded network. Upon transition to the open state (red), this network gets interrupted: K940 bends towards T978 and D407 reorients towards the periplasm. Additionally, the R971 side chain relocates in the direction of the cytoplasm. B) During the transition from tight to open state (blue to red), there is a coil-to-helix transition at the N-terminal end of TM8. Concomitantly, the PC2 subdomain shifts towards the membrane plane by 6.5 Å. The N-terminal end of helix 8 and the C-terminal β-strand of the PC2 subdomain are shown. C) Top view of the triad from the cytoplasm. In addition to the drastic reorientation of K940, a bulging of TM5 towards TM4 and TM10 can be observed. The pictures were generated by superimposition of the loose and the open monomer. The pdb coordinate file 2GIF was used for figures A) and B), and 2J8S for figure C).
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Publications
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Seeger, M. A., von Ballmoos, C., Verrey, F. and Pos, K.M. (2009) Crucial role of Asp408 in the proton translocation pathway of multidrug transporter AcrB: Evidence from site-directed mutagenesis and carbodiimide labeling. Biochemistry 48, 5801-5812.
Pos, K. M. (2009) Drug transport mechanism of the AcrB efflux pump. Biochim Biophys Acta 1794, 782-793.
Bohnert J. A., Schuster S., Seeger M.A., Fähnrich E., Pos K.M. and Kern W.V (2008) Site-Directed Mutagenesis reveals Putative Substrate Binding Residues in the Escherichia coli RND Efflux Pump AcrB. J Bacteriol 190, 8225–8229.
Seeger, M. A., von Ballmoos, C., Eicher, T., Brandstätter, L., Verrey, F., Diederichs, K. and Pos, K. M. (2008) Engineered disulfide bonds support the functional rotation mechanism of multidrug efflux pump AcrB. Nature Struc Mol Biol 15, 199-205.
Seeger, M. A., Schiefner, A., Eicher, T., Verrey, F., Diederichs, K. and Pos, K. M. (2006) Asymmetric structure of trimeric AcrB suggests a peristaltic drug pumping mechanism. Science 313, 1295-1298.