This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Bacteria such as Escherichia coli have developed various mechanisms to overcome toxic environments that are otherwise unfavorable for their survival. One important strategy that bacteria use to subvert toxic compounds, including heavy metal ions, is the expression of membrane transporters that recognize and actively export these toxic compounds out of bacterial cells, thereby allowing the bugs to survive in extremely toxic conditions. Our long-term goal is to elucidate the structures and fundamental mechanisms that give rise to heavy metal ion recognition and extrusion in heavy metal efflux proteins. The primary target of this proposal is the E. coli CusABC efflux system that recognizes and extrudes silver and copper ions out of the bacterial cell. CusA consists of 1,047 amino acid residues. It is an inner membrane transporter, which belongs to the resistance-nodulation-division (RND) protein superfamily. CusC is a 457 amino acid polypeptide that forms an outer membrane channel in E. coli. These two membrane proteins interact with each other, in conjunction with a membrane fusion protein CusB (379 amino acids), to mediate the extrusion of heavy metal ions across both membranes of E. coli. It has been proposed that CusB may act as an adaptor that brings CusA and CusC together to form the CusABC tripartite complex. This efflux complex makes direct contact with the metal ions and selectively expels them out of the cell. We recently cloned, expressed, and purified the full-length CusA, CusC, and CusB efflux proteins. We also crystallized each protein in detergent solution using vapor-diffusion. X-ray diffraction data were collected from cryocooled crystals at a synchrotron light source.
The specific aims are to determine the structural basis of heavy-metal ion interactions with: (1) the CusA inner membrane efflux pump, (2) the CusC outer membrane channel, and (3) the CusB membrane fusion protein.
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