Not Just a Passive Adaptor, the Periplasmic Component CusB of Escherichia coli's CusCFBA Copper Efflux System has an Active Functional Role

Abstract

Increased emergence of antibiotic resistance in bacterial pathogens has posed a serious threat to human health. Due to similar structural and functional characteristics of metal and antibiotic resistance systems in gram-negative bacteria, there is a growing concern that metal contamination functions as a selective agent in the proliferation of antibiotic resistance. The CusCFBA copper/silver resistance system of Escherichia coli forms a protein complex that spans the inner and outer membranes and functions in the efflux of metal from the periplasm to the extracellular space. In order to understand the molecular details of metal resistance by the Cus system and more specifically, to define the role of the periplasmic components in CBA type metal transporters, I characterized CusB and probed its interactions with CusF using various structural and biochemical tools. CusB was previously thought to play a relatively passive role as an adaptor protein that stabilized the association of the inner and outer membrane proteins. Through isothermal titration calorimetry (ITC), X-ray absorption spectroscopy (XAS), and mutagenesis, I have shown that CusB binds Cu(I)/Ag(I) with high affinity using three conserved methionines. Gel filtration chromatography experiments showed that upon binding Ag(I), CusB undergoes a substantial conformational change. Importantly, functional metal binding by CusB is essential for cell survival in environments with elevated copper concentrations. The small periplasmic metal binding protein CusF is a unique component of monovalent metal resistance systems serving an unknown function. To determine the nature and specificity of interaction between CusF and CusB, ITC and NMR were used to show that the interaction between CusF and CusB is metal-dependent and specific for the components of Cus system. From NMR chemical shift perturbations, the CusB interaction face on CusF was determined to overlap with the metal binding site. XAS experiments demonstrate metal transfer between CusB and CusF, which supports the role of CusF as a metallochaperone. In summary, these findings demonstrate an active role for CusB in metal resistance, and suggest that the plausible role for CusF is that of a metallochaperone for CusB.