Structural and Functional Investigation of the Ag+/Cu+ Binding Domains of the Periplasmic Adaptor Protein SilB from Cupriavidus metallidurans CH34

Silver ion resistance in bacteria mainly relies on efflux systems, and notably on tripartite efflux complexes involving a transporter from the resistance–nodulation–cell division (RND) superfamily, such as the SilCBA system from Cupriavidus metallidurans CH34. The periplasmic adaptor protein SilB hosts two specific metal coordination sites, located in the N-terminal and C-terminal domains, respectively, that are believed to play a different role in the efflux mechanism and the trafficking of metal ions from the periplasm to the RND transporter. On the basis of the known domain structure of periplasmic adaptor proteins, we designed different protein constructs derived from SilB domains with either one or two metal binding sites per protein chain. ITC data acquired on proteins with single metal sites suggest a slightly higher affinity of Ag+ for the N-terminal metal site, compared to that for the C-terminal one. Remarkably, via the study of a protein construct featuring both metal sites, nuclear magnetic re...