A New Critical Conformational Determinant of Multidrug Efflux by an MFS Transporter |
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Authors: | Elia Zomot Eliane Hadas Yardeni Attilio Vittorio Vargiu Heng-Keat Tam Giuliano Malloci Venkata Krishnan Ramaswamy Michal Perach Paolo Ruggerone Klaas Martinus Pos Eitan Bibi |
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Institution: | 1. Department of Biomolecular Sciences, Weizmann Institute of Science, Rehovot 76100, Israel;2. Department of Physics, University of Cagliari, 09042 Monserrato (CA), Italy;3. Institute of Biochemistry, Goethe University Frankfurt, 60438 Frankfurt, Germany |
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Abstract: | Secondary multidrug (Mdr) transporters utilize ion concentration gradients to actively remove antibiotics and other toxic compounds from cells. The model Mdr transporter MdfA from Escherichia coli exchanges dissimilar drugs for protons. The transporter should open at the cytoplasmic side to enable access of drugs into the Mdr recognition pocket. Here we show that the cytoplasmic rim around the Mdr recognition pocket represents a previously overlooked important regulatory determinant in MdfA. We demonstrate that increasing the positive charge of the electrically asymmetric rim dramatically inhibits MdfA activity and sometimes even leads to influx of planar, positively charged compounds, resulting in drug sensitivity. Our results suggest that unlike the mutants with the electrically modified rim, the membrane-embedded wild-type MdfA exhibits a significant probability of an inward-closed conformation, which is further increased by drug binding. Since MdfA binds drugs from its inward-facing environment, these results are intriguing and raise the possibility that the transporter has a sensitive, drug-induced conformational switch, which favors an inward-closed state. |
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Keywords: | MdfA multidrug resistance multidrug transport secondary transport major facilitator superfamily Mdr multidrug MFS major facilitator superfamily Cm chloramphenicol Eth ethidium TPP tetraphenylphosphonium Dq dequalinium Acr acriflavine DXC deoxycholate MD molecular dynamics CL cysteine-less β-Me β-mercaptoethanol DDM CCCP carbonyl cyanide 3-chlorophenylhydrazone POPE 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine |
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