Intracellular copper levels must be tightly regulated to avoid toxicity yet meet the copper requirements of the cell. The CBA efflux system CusCFBA is a copper resistance system of Escherichia coli, which is especially important under anaerobic conditions. The cus determinant contains an additional component, the periplasmic protein CusF, which is not found in other CBA efflux systems. We have determined the crystal structure of apo-CusF. The protein forms a five-stranded β-barrel, classified as an OB-fold, which is a unique topology for a copper-binding protein. NMR chemical shift mapping experiments as well as EXAFS data suggest that Cu(I) is bound by conserved residues H36, M47, and M49 located in β-strands 2 and 3. 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. NMR and ITC have shown that CusF also binds Ag(I) with high affinity. We have solved the crystal structure of the CusF-Ag(I) complex. CusF binds Ag(I) at the site also proposed for Cu(I) binding and with the same ligands, H36, M47 and M49. Upon binding of Ag(I) the protein undergoes a slight structural rearrangement, which may trigger protein-protein interactions necessary for optimal copper/silver resistance. OB-fold proteins have a common binding face, which maps to a highly conserved region in CusF and its homologs and to the same region on CusF that undergoes structural changes upon metal binding. We are in the process of determining if this conserved region is used by CusF to interact with other proteins, using in vivo crosslinking to identify possible interaction partners of CusF and thus hope to clarify the function of CusF within the Cus system.