Gram-negative bacteria utilize dual membrane resistance nodulation division-type efflux systems to export a variety of substrates. These systems contain an essential periplasmic component that is important for assembly of the protein complex. We show here that the periplasmic protein CusB from the Cus copper/silver efflux system has a critical role in Cu(I) and Ag(I) binding. Isothermal titration calorimetry experiments demonstrate that one Ag(I) ion is bound per CusB molecule with high affinity. X-ray absorption spectroscopy data indicate that the metal environment is an all-sulfur 3-coordinate environment. Candidates for the metal-coordinating residues were identified from sequence analysis, which showed four conserved methionine residues. Mutations of three of these methionine residues to isoleucine resulted in significant effects on CusB metal binding in vitro. Cells containing these CusB variants also show a decrease in their ability to grow on copper-containing plates, indicating an important functional role for metal binding by CusB. Gel filtration chromatography demonstrates that upon binding metal, CusB undergoes a conformational change to a more compact structure. Based on these structural and functional effects of metal binding, we propose that the periplasmic component of resistance nodulation division-type efflux systems plays an active role in export through substrate-linked conformational changes.Efflux systems of the resistance nodulation division (RND) 2 family are key players in the intrinsic and acquired antibiotic resistance of Gram-negative bacteria (1). These systems confer resistance to otherwise lethal concentrations of drugs and metal ions, and they also mediate efflux of bacterial products such as siderophores, peptides, and quorum-sensing signals (2, 3). With antibiotic-resistant pathogens representing a growing threat to human health, understanding these efflux systems is of significant importance.RND-type efflux systems form a transenvelope complex comprised of three fundamental components: an energy-utilizing inner membrane protein (4), an outer membrane factor, and a periplasmic component (5). The inner membrane components are proton-substrate antiporters of the RND protein superfamily, which are subclassified on the basis of their exported substrate (4). Members of the heavy metal efflux subfamily of RND transport systems are highly substrate-specific, with the ability to differentiate between monovalent and divalent ions (4). In contrast, the hydrophobe/amphiphile efflux (HAE) subfamily of RND protein systems has significantly broader substrate recognition. Members of the HAE-RND systems transport a wide range of structurally unrelated molecules, including antibiotics, dyes, detergents, bile salts, organic solvents, and antimicrobial peptides (6).Insights into the functions of the three fundamental components of RND efflux systems have been gathered from studies of a variety of RND systems. By far, the most information at the structural and biochemical levels is known for the inn...

We have determined the crystal structure of apo-CusF, a periplasmic protein involved in copper and silver resistance in Escherichia coli. The protein forms a five-stranded beta-barrel, classified as an OB-fold, which is a unique topology for a copper-binding protein. NMR chemical shift mapping experiments suggest that Cu(I) is bound by conserved residues H36, M47, and M49 located in beta-strands 2 and 3. These residues are clustered at one end of the beta-barrel, and their side chains are oriented toward the interior of the barrel. Cu(I) can be modeled into the apo-CusF structure with only minimal structural changes using H36, M47, and M49 as ligands. The unique structure and metal binding site of CusF are distinct from those of previously characterized copper-binding proteins.

Transition metals require exquisite handling within cells to ensure that cells are not harmed by an excess of free metal species. In gram-negative bacteria, copper is only required in low amounts in the periplasm, not in the cytoplasm, so a key aspect of protection under excess metal conditions is to export copper from the periplasm. Additional protection could be conferred by a periplasmic chaperone in order to limit the free metal species prior to export. Using isothermal titration calorimetry, we have demonstrated that two periplasmic proteins, CusF and CusB, of the E. coli Cu (I)/Ag(I) efflux system undergo a metal dependent interaction. Through the development of a novel X-ray absorption spectroscopy approach using selenomethionine labeling to distinguish the metal sites of the two proteins, we have demonstrated transfer of Cu(I) occurs between CusF and CusB. The interaction between these proteins is highly specific, as a homolog of CusF with 51% sequence identity and similar affinity for metal, did not function in metal transfer. These experiments establish a metallochaperone activity for CusF in the periplasm of gram-negative bacteria, serving to protect the periplasm from metal-mediated damage.

Elevated levels of copper or silver ions in the environment are an immediate threat to many organisms. Escherichia coli is able to resist the toxic effects of these ions through strictly limiting intracellular levels of Cu(I) and Ag(I). The CusCFBA system is one system in E. coli responsible for copper/silver tolerance. A key component of this system is the periplasmic copper/silver-binding protein, CusF. Here the X-ray structure and XAS data on the CusF-Ag(I) and CusF-Cu(I) complexes, respectively, are reported. In the CusF-Ag(I) structure, Ag(I) is coordinated by two methionines and a histidine, with a nearby tryptophan capping the metal site. EXAFS measurements on the CusF-Cu(I) complex show a similar environment for Cu(I). The arrangement of ligands effectively sequesters the metal from its periplasmic environment and thus may play a role in protecting the cell from the toxic ion.Keywords: metal tolerance; copper; silver; metal coordination; crystallography; X-ray absorption spectroscopy Supplemental material: see www.proteinscience.orgThe intracellular concentrations of metals must be carefully regulated to avoid toxic effects. One way in which Escherichia coli responds to elevated levels of copper or silver in its environment is through the up-regulation of the cusCFBA operon (Munson et al. 2000;Franke et al. 2001). Three of the proteins encoded by this operon, CusCBA, are expected to form an efflux complex spanning the periplasm, similar to the well-characterized multidrug transporters (Franke et al. 2003). However, the fourth component of this system, CusF, is unique to copper/silver transport systems and is essential for the Cus system to achieve its maximal function (Franke et al. 2003). CusF shows high affinity for both Cu(I) and Ag(I), which have similar properties; however, it does not appreciably bind Cu(II) (Kittleson et al. 2006). Homologs of this small periplasmic metal-binding protein are present in all putative copper/silver tolerance systems, yet its function within these systems has not yet been described. CusF may act as a metallochaperone and be involved in metal tolerance through selection of metal substrates or it may regulate the efflux complex through protein-protein interactions. To further describe its role Abbreviations: rmsd, root mean square deviation; XAS, X-ray absorption spectroscopy DW: Debye-Waller.Article and publication are at

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Bacteria have evolved several transport mechanisms to maintain metal homeostasis and to detoxify the cell. One mechanism involves an RND (resistance-nodulation-cell division protein family)-driven tripartite protein complex to extrude a variety of toxic substrates to the extracellular milieu. These efflux systems are comprised of a central RND proton-substrate antiporter, a membrane fusion protein, and an outer membrane factor. The mechanism of substrate binding and subsequent efflux has yet to be elucidated. However, the resolution of recent protein crystal structures and genetic analyses of the components of the heavy-metal efflux family of RND proteins have allowed the developments of proposals for a substrate transport pathway. Here two models of substrate extrusion through RND protein complexes of the heavy-metal efflux protein family are described. The funnel model involves the shuttling of periplasmic substrate from the membrane fusion protein to the RND transporter and further on through the outer membrane factor to the extracellular space. Conversely, the switch model requires substrate binding to the membrane fusion protein, inducing a conformational change and creating an open-access state of the tripartite protein complex. The extrusion of periplasmic substrate bypasses the membrane fusion protein, enters the RND-transporter directly via its substrate-binding site, and is ultimately eliminated through the outer membrane channel. Evidence for and against the two models is described, and we propose that current data favor the switch model.In Gram-negative bacteria, RND-driven tripartite protein complexes pump out a wide array of substrates. The RND (resistance-nodulation-cell division) protein superfamily can be divided into seven protein families with members being involved in transport of organic substances, transition metals, and polypeptides (55). Two RND families have been most extensively studied. The hydrophobe/amphiphile efflux family includes the RND proteins AcrB from Escherichia coli and MexB from Pseudomonas aeruginosa, both of which are involved in export of organic substances such as various antibiotics, bile salts, and other hydrophobic substances. The heavy metal efflux family contains metal transporters, such as CzcA from Cupriavidus metallidurans strain CH34 and CusA from E. coli. The nonspecificity of the RND proteins exporting organic substances has made elucidation of the actual transport pathway and pump regulation difficult. In contrast, metal-transporting RND proteins have a limited substrate spectrum, pumping out either the monovalent cations Cu(I) and Ag(I) or divalent cations of the transition metals Zn(II), Ni(II), Co(II), and sometimes the heavy metal cation Cd(II) (9, 25, 33). Since metals bind to very specific residues, the elucidation of the transport pathway should be easier.RND proteins utilize the proton motive force to drive the efflux of the substrates (14, 32). They form homotrimers (29) or heterotrimers composed of two different RND polypeptides in a 2:1 ratio (18...

Background: It is unknown how soluble chaperones acquire Cu ϩ for delivery to metalloproteins and transporters.

Copper is an essential nutrient for all aerobic organisms but is toxic in excess. At the host-pathogen interface, macrophages respond to bacterial infection by copper-dependent killing mechanisms, whereas the invading bacteria are thought to counter with an up-regulation of copper transporters and efflux pumps. The tripartite efflux pump CusCBA and its metallochaperone CusF are vital to the detoxification of copper and silver ions in the periplasm of Escherichia coli. However, the mechanism of efflux by this complex, which requires the activation of the inner membrane pump CusA, is poorly understood. Here, we use selenomethionine (SeM) active site labels in a series of biological X-ray absorption studies at the selenium, copper, and silver edges to establish a "switch" role for the membrane fusion protein CusB. We determine that metal-bound CusB is required for activation of cuprous ion transfer from CusF directly to a site in the CusA antiporter, showing for the first time (to our knowledge) the in vitro activation of the Cus efflux pump. This metal-binding site of CusA is unlike that observed in the crystal structures of the CusA protein and is composed of one oxygen and two sulfur ligands. Our results suggest that metal transfer occurs between CusF and apo-CusB, and that, when metal-loaded, CusB plays a role in the regulation of metal ion transfer from CusF to CusA in the periplasm.copper | periplasmic efflux | X-ray absorption spectroscopy | metal ion transport | host-pathogen interaction