A copper active site in the periplasmic efflux transporter protects gram-negative bacteria

The spread out of resistant pathogenic microbes become one of the most challenging problems that modern medicine faces. Development of novel drugs based on new molecular targets, which were not targeted yet, is of the highest priority in antibiotic research. One approach that has been suggested recently is to inhibit copper transporters in prokaryotic systems. Copper is required for many biological pathways, however, can be deleterious to the cell. Pathogenic systems hold highly sophisticated copper regulation network. Understanding at the molecular level how this network operates, will assist in development the next generation of antibiotics. CusB protein is part of the E.coli CusCFBA periplasmic Cu(I) efflux system, which is part of the gram-negative bacteria. Lately, it was reported that CusB plays a key role in opening of the whole CusCFBA system. Where both conformational changes as well as assembly/disassembly process control the opening of the transporter. The missing link in getting a full understanding on the CusB function is targeting the various Cu(I) binding sites in CusB, and identifying an essential active site in CusB. Then, using this site, a new inhibitor to CusB can be later designed. This is the focus of this research. Here, we combine both in vitro structural measurements, as well as cell experiments to identify the active site in CusB. We reveal that this site involves two important methionine residues: M227 and M241, which are located near a solvent exposed area between domain 2 and domain 3 of CusB. Mutations of these residues, result in nonfunctional CusB protein, and decrease copper resistance.