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.

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

The periplasmic protein CusF, as a part of the CusCFBA efflux complex, plays a role in resistance to elevated levels of copper and silver in Escherichia coli. Although homologues have been identified in other Gram-negative bacteria, the substrate of CusF and its precise role in metal resistance have not been described. Here, isothermal titration calorimetry (ITC) was used to demonstrate that CusF binds with high affinity to both Cu(I) and Ag(I) but not Cu(II). The affinity of CusF for Ag(I) was higher than that for Cu(I), which could reflect more efficient detoxification of Ag(I) given the lack of a cellular need for Ag(I). The chemical shifts in the nuclear magnetic resonance (NMR) spectra of CusF-Ag(I) as compared to apo-CusF show that the region of CusF most affected by Ag(I) binding encompasses three absolutely conserved residues: H36, M47, and M49. This suggests that these residues may play a role in Ag(I) coordination. The NMR spectra of CusF in the presence of Cu(II) do not indicate specific binding, which is in agreement with the ITC data. We conclude that Cu(I) and Ag(I) are the likely physiological substrates.

The periplasmic metallochaperone CusF coordinates Cu(I) and Ag(I) through a unique site consisting of a Met 2 His motif as well as a Cu(I)-π interaction between a nearby tryptophan, W44, and the metal ion. Through mutational analyses we investigate here the role that W44 in CusF plays in metal coordination. Nuclear magnetic resonance spectra show that the specificity of CusF for Cu(I) and Ag(I) is not altered by mutation of W44. X-ray absorption studies reveal that W44 protects the bound Cu(I) from oxidation as well as from adventitious ligands. Competition assays demonstrate that W44 does not significantly contribute to the affinity of CusF for metal, but that substitution of W44 by methionine, which forms a fourth Cu(I) ligand, substantially increases the affinity. These studies indicate that W44 is important in maintaining a moderate-affinity and solvent-shielded threecoordinate environment for Cu(I), which has implications for the function of CusF as a metallochaperone.

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