Crucial role of Asp408 in the proton translocation pathway of multidrug transporter AcrB: evidence from site-directed mutagenesis and carbodiimide labeling.
The three-component AcrA/AcrB/TolC efflux system of Escherichia coli catalyzes the proton motive force-driven extrusion of a variety of cytotoxic compounds. The inner membrane pump component AcrB belongs to the resistance nodulation and cell division (RND) superfamily and is responsible for drug specificity and energy transduction of the entire tripartite efflux system. Systematic mutational analysis of titratable and polar membrane-located amino acids revealed four residues, D407, D408, K940, and, R971, to be of prime importance for AcrB function. Using matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry, D408 was shown to specifically react with dicyclohexylcarbodiimide (DCCD) in a pH-dependent manner. The apparent pK(a) of D408 of 7.4 would enable binding and release of protons under physiological conditions. In contrast to other secondary transporters, D408 was not protected from carbodiimide modification in the presence of drugs, which supports the notion of spatially separated transport pathways for drugs and protons. This study provides evidence for a substantial role of membrane-located carboxylates as a central element of the proton translocation pathway in AcrB and other members of the RND superfamily.
Institute of Physiology and Zurich Centre for Integrative Human Physiology (ZIHP), University of Zurich, Winterthurerstrasse 190, CH-8057 Zurich, Switzerland.
Markus A
MA
Seeger
Christoph
C
von Ballmoos
François
F
Verrey
Klaas M
KM
Pos
D000595:Amino Acid Sequence; D001224:Aspartic Acid / Q000235:genetics* / Q000378:metabolism / Q000502:physiology*; D002234:Carbodiimides / Q000378:metabolism*; D002352:Carrier Proteins / Q000378:metabolism / Q000502:physiology; D002455:Cell Division / Q000235:genetics; D029968:Escherichia coli Proteins / Q000235:genetics* / Q000378:metabolism / Q000502:physiology*; D008969:Molecular Sequence Data; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D005810:Multigene Family; D016297:Mutagenesis, Site-Directed / Q000379:methods; D006180:Proton-Translocating ATPases / Q000235:genetics* / Q000378:metabolism / Q000502:physiology*; D015398:Signal Transduction / Q000235:genetics; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013194:Staining and Labeling / Q000379:methods
D001224:Aspartic Acid; D002234:Carbodiimides; D002352:Carrier Proteins; D029968:Escherichia coli Proteins; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D006180:Proton-Translocating ATPases; C083496:AcrB protein, E coli; C027224:carbodiimide binding proteins