Energetics and Topology of CzcA, a Cation/Proton Antiporter of the Resistance-Nodulation-Cell Division Protein Family

Abstract

The membrane-bound CzcA protein, a member of the resistance-nodulation-cell division (RND) permease superfamily, is part of the CzcCB 2 A complex that mediates heavy metal resistance in Ralstonia sp. CH34 by an active cation efflux mechanism driven by cation/proton antiport. CzcA was purified to homogeneity after expression in Escherichia coli, reconstituted into proteoliposomes, and the kinetics of heavy metal transport by CzcA was determined. CzcA is composed of 12 transmembrane ␣-helices and two large periplasmic domains. Two conserved aspartate and a glutamate residue in one of these transmembrane spans are essential for heavy metal resistance and proton/cation antiport but not for facilitated diffusion of cations. Generalization of the resulting model for the function of CzcA as a two-channel pump might help to explain the functions of other RND proteins in bacteria and eukaryotes.Multiple drug resistant bacteria poses a threat to man's fight against infectious diseases. Some multiple drug resistance systems may detoxify their substrates by transport across the complete cell wall of Gram-negative bacteria, across cytoplasmic membrane, periplasm, and outer membrane. These assumed transenvelope transporters are composed of a pump protein that energizes the transport, in addition to a membrane fusion and an outer membrane-associated protein (1, 2). The pump protein may be an ATP-binding cassette transporter (3, 4), a transporter of the major facilitator superfamily (5), or a resistance-nodulation-cell division (RND) 1 protein (4, 6, 7). The archetype of the RND permease superfamily family is CzcA from the Gram-negative bacterium Ralstonia sp. CH34 (formerly Alcaligenes eutrophus strain CH34) (8 -12).This bacterium contains at least seven heavy metal resistance determinants, located either on the bacterial chromosome or on one of the two indigenous plasmids pMOL28 (163 kilobase pairs) and pMOL30 (238 kb) (8, 13-16). One of them, the czc-determinant of plasmid pMOL30, mediates inducible resistance to millimolar concentrations of Co 2ϩ , Zn 2ϩ , and Cd 2ϩ in strain CH34 (8, 17). The products of the genes czcA, czcB, and czcC form a membrane-bound protein complex catalyzing an energy-dependent efflux of these three metal cations (9, 11), probably across the complete envelope. The mechanism of action of CzcCB 2 A is that of a proton/cation antiporter, and the K m values of the efflux system for the substrate heavy metal cations are also in the millimolar range (10). Although indirect evidence led to the assumption that CzcA is the central cation/proton antiporter of the CzcCB 2 A complex (10), this has not been shown directly. This paper demonstrates that CzcA is a cation/proton antiporter, and develops the model of CzcA as a two-channel pump based on topology studies and the function of CzcA mutant proteins. This model sheds some light on other RND proteins involved in multiple drug resistance of bacteria or with previously unknown functions in mammals. EXPERIMENTAL PROCEDURESBacterial Strains, Plasmids, and Growt...

Kinetic behavior of the major multidrug efflux pump AcrB of Escherichia coli

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Amino Acid Residues Essential for Function of the MexF Efflux Pump Protein of Pseudomonas aeruginosa

“…The E417K substitution yielded a normally expressed MexF protein (Fig. 2) (5). Since the drugtransporting pumps Acr and Mex are able to extrude uncharged hydrophobic molecules like organic solvents and chloramphenicol, it was proposed that a charge relay system was not involved in drug/proton translocation (5, 31).…”

Evidence for a role of vertebrate Disp1 in long-range Shh signaling

“…Disp1 and Ptch1 share many characteristics with members of the resistance-nodulation-cell division (RND) family of protondriven transporters. Not only are the overall structures of Disp1 and Ptch1 similar to that of bacterial RND permeases, but aspartic acid residues in the fourth transmembrane span, which are thought to be important for proton translocation, are also conserved (Goldberg et al, 1999;Tseng et al, 1999). It has previously been shown that bacterial RND permeases function as trimers (Nagano et al, 2005).…”