Identification of essential charged residues in transmembrane segments of the multidrug transporter MexB of Pseudomonas aeruginosa

The MexABM efflux pump exports structurally diverse xenobiotics, utilizing the proton electrochemical gradient to confer drug resistance on Pseudomona s aeruginosa. The MexB subunit traverses the inner membrane 12 times and ha s two, two, and one charged residues in putative transmembrane segments 2 ( TMS-2), TMS-4, and TMS- 10, respectively. All five residues were mutated, a nd MexB function was evaluated by determining the MICs of antibiotics and f luorescent dye efflux, Replacement of Lys342 with Ala, Arg, or Glu and Glu3 46 with Ala, GIn, or Asp in TMS-2 did not have a discernible effect. Ala, A sn, or Lys substitution for Asp407 in TMS-4, which is well conserved, led t o loss of activity. Moreover, a mutant with Glu in place of Asp407 exhibite d only marginal function, suggesting that the length of the side chain at t his position is important, The only replacements for Asp408 in TMS-4 or Lys 939 in TMS-10 that exhibited significant function were Glu and Arg, respect ively, suggesting that the native charge at these positions is required. In addition, double neutral mutants or mutants in which the charged residues Asp407 and Lys939 or Asp408 and Lys939 were interchanged completely lost fu nction, An Asp408->Glu/Lys939->Arg mutant retained significant activity, wh ile an Asp407->Glu/Lys939->Arg mutant exhibited only marginal function. An Asp407->Glu/ Asp4408->Glu double mutant also lost activity but significant function was restored by replacing Lys939 with Arg (Asp407->Glu/Asp408->Glu /Lys939->Arg). Taken as a whole, the findings indicate that Asp407, Asp408, and Lys939 are functionally important and raise the possibility that Asp40 7, Asp408, and Lys939 may form a charge network between TMS-4 and TMS-10 th at is important for proton translocation and/or energy coupling.