The MexB subunit of the MexABM efflux pump exports an extremely broad range of substrates, including stereochemically and electrically unrelated xenobiotics. The only properties shared by the substrate compounds are hydrophobicity and amphiphilicity (16
). Phylogenic analysis showed that most RND transporters have a transmembrane topology similar to that of MexB and extrude the substrate utilizing the proton motive force (5
). Recent in vitro reconstitution studies of two members of this family have confirmed that RND transporters are proton-substrate antiporters (4
). However, the mechanism by which the pump couples cellular energy, substrate selection, and transport remained elusive.
We ran a multiple alignment of the full-length amino acid sequences of 39 proteins that belong to the heavy metal efflux and HAE-1 subsets of the RND superfamily. G403XXXD407XXXXXXE414 in TMS-4 of MexB is strictly conserved in all of the sequences aligned. These residues are predicted to line up on the same face of the α-helical TMS, and their strict conservation suggests that they play an important role(s) in a proton translocation that is shared by the family of proteins (Fig. ). D408 in TMS-4 and K939 in TMS-10 in the MexB protein are only conserved in the HAE-1 family.
Ala replacement at position 407, 408, or 939 inactivated the pump function without decreasing the expression of mutant proteins, indicating that these conserved charges located in the central region of TMS play important roles in pump function. Highly conserved residue E414 is two turns away from D407 and may be located at the boundary of the cytoplasmic side of TMS-4. This residue might also play an important role and should be further investigated. Substitutions of Asn or Lys for D407 and D408 lead to complete loss of extrusion activity. Replacement of K939 with Glu or Asp also inactivated pump function. D407 could not be replaced with Glu, in which the carboxyl-containing side chain is one methylene longer, suggesting that side-chain length at position 407 is important. Pump function was preserved when D408 was replaced with Glu or K939 was replaced with Arg, suggesting that positions 408 and 939 required a negative and a positive charge, respectively. The volume of the side chain may not be essential. The fact that these two positions could tolerate larger side chains also suggested that TMS-4 and TMS-10 are flexible. It was reported that D408 of Ralstonia eutropha
CzcA, a member of the RND family, corresponding to D407 of MexB, is essential for proton translocation (4
). The role of D407 in MexB might be similar to that of D408 in CzcA of R. eutropha
. Thus, it is likely that conserved D407, D408, and K939 in MexB are involved in proton translocation and/or energy coupling.
The D408E/K939R double mutant, where the charge pair was conserved, showed significant pump function, while the D407E/K939R mutant exhibited only marginal function, similar to that of the D407E single mutant. The D407E/D408E double mutant totally lost pump function, but significant activity was restored by replacing K939 with Arg. The D407A/K939A and D408A/K939A double neutral mutants and mutants in which charged residues D407 and K939 or D408 and K939 were interchanged also totally lost pump function. All of these data support the idea that D407, D408, and K939 play an important role(s) in pump function.
Amphiphilic TMS-4 and TMS-10 contained important charged residues, two negative charges in TMS-4, and one positive charge plus one polar residue (N940) in TMS-10. It is possible that these important charges form ionic interactions to stay in the hydrophobic membrane environment. There is no functional restoration upon charge reversal substitution or double neutral mutation. Given that three charges form a charge network, all of the experimental data are not against this assumption. The use of approaches such as site-directed cysteine cross-linking or engineered divalent metal binding sites in conjunction with electron paramagnetic resonance would be a way to study the geometrical relationship between these sites and the interaction of TMS-4 and TMS-10 (7