Nadine Fischer

Publications (2)8.05 Total impact

• Article: Porter Domain Opening and Closing Motions in the Multi-Drug Efflux Transporter AcrB.

  Nadine Fischer, Christian Kandt
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  ABSTRACT: AcrB acts as the active transporter in the multi-drug efflux pump AcrAB-TolC in Escherichia coli. Within the same reaction cycle intermediate all AcrB X-ray structures display highly similar conformations of the substrate-recruiting and transporting porter domain. To assess if this structural homogeneity is an intrinsic feature of AcrB or stems from other causes we performed a series of six independent, unbiased 100ns molecular dynamics simulations of membrane-embedded, asymmetric, substrate-free wild type AcrB in a 150mM NaCl solution. We find the porter domain more flexible than previously assumed displaying clear opening and closing motions of the proximal binding pocket (L and T-state) and the exit of the drug transport channels (O-intermediate). Concurrently the hydrophobic binding pocket favors a closed conformation in all three protomers. Our findings suggest that the conformational homogeneity seen in the crystal structures is likely an effect of bound but structurally unresolved substrate. Our simulations further imply that each of the known three reaction cycle intermediates occurs in at least two variants, the Thr676 loop independently regulates porter domain access and likely plays a key role in substrate transport. On a 100ns time scale we find no evidence supporting the proposed LLL resting state in the absence of substrate. If the proximal binding pocket dynamics have an inhibiting effect on AcrB pump activity lowering the life time of substrate-accessible conformations, the observed dynamics could provide a structural explanation for the AcrB activity-enhancing effect of the adaptor protein AcrA stabilizing PC1 and PC2 subdomain orientations.
  Biochimica et Biophysica Acta 10/2012; · 4.66 Impact Factor
• Article: Three ways in, one way out: water dynamics in the trans-membrane domains of the inner membrane translocase AcrB.

  Nadine Fischer, Christian Kandt
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  ABSTRACT: Powered by proton-motive force, the inner membrane translocase AcrB is the engine of the AcrAB-TolC efflux pump in Escherichia coli. As proton conduction in proteins occurs along hydrogen-bonded networks of polar residues and water molecules, knowledge of the protein-internal water distribution and water-interacting residues allows drawing conclusions to possible pathways of proton conduction. Here, we report a series of 6× 50 ns independent molecular dynamics simulations of asymmetric AcrB embedded in a phospholipid/water environment. Simulating each monomer in its proposed protonation state, we calculated for each trans-membrane domain the average water distribution, identified residues interacting with these waters and quantified each residue's frequency of water hydrogen bond contact. Combining this information we find three possible routes of proton transfer connecting a continuously hydrated region of known key residues in the TMD interior to bulk water by one cytoplasmic and up to three periplasm water channels in monomer B and A. We find that water access of the trans-membrane domains is regulated by four groups of residues in a combination of side chain re-orientations and shifts of trans-membrane helices. Our findings support a proton release event via Arg971 during the C intermediate or in the transition to A, and proton uptake occurring in the A or B state or during a so far unknown intermediate in between B and C where cytoplasmic water access is still possible. Our simulations suggest experimentally testable hypotheses, which have not been investigated so far.
  Proteins Structure Function and Bioinformatics 10/2011; 79(10):2871-85. · 3.39 Impact Factor