Structural Studies of Copper‐Binding Proteins Encoded by the ycnOperon from Bacillus subtilis

Abstract

Most living organisms encode an ensemble of protein transporters and chaperones to regulate copper levels. The ycn operon of the bacterium Bacillus subtilisis regulated in response to copper, suggesting that the three proteins it encodes may engage in direct interactions with copper ions to facilitate transport and homeostasis of this transition metal. Here, we use a combination of structural, biochemical, biophysical, and bioinorganic approaches to determine the structures of individual proteins and their interactions with copper and other binding partners. The YcnI protein encoded by the operon contains a domain of unknown function (DUF1775) at its N‐terminus. We determined the structure of this domain and characterized its interactions with copper ions, finding that it coordinates the metal using a unique “mono‐histidine brace” site that is highly conserved across other members of the DUF1775 family. Our data also suggest a potential role for this domain to serve as a chaperone to facilitate transfer of metal ions. Furthermore, we also identify potential copper binding sites within the transcriptional regulator encoded within the same operon. Together, these studies reveal new insights into the structure and copper‐binding properties of proteins encoded by the ycnoperon, providing additional perspectives on how they may work together to regulate copper homeostasis. As homologs of these proteins are shared by many other bacterial species, it is likely that analogous mechanisms exist for copper uptake and homeostasis beyond B. subtilis.