All organisms require metals as co-factors, and these essential metals must be obtained from external sources. Gram- negative bacteria possess specialized pathways that enable binding and transport across three cellular compartments: the outer membrane, the periplasm and the inner membrane. The outer membrane contains transporters specific for different metals or metal-containing compounds; in pathogenic bacteria, these transporters are implicated as virulence factors. As the outer membrane does not maintain an electrochemical gradient, energy- dependent steps in transport across the outer membrane are mediated through proteins that couple the inner membrane gradient to the outer membrane transporter. These bacterial systems provide a tractable model for a molecular dissection of active transport. We seek to understand the mechanism of these transport processes through crystallographic and structure/function studies of bacterial cobalamin transport. We have obtained three-dimensional crystals of the Escherichia coli vitamin cobalamin transporter BtuB that diffract to beyond 2.0 Angstrom units resolution (2.1 Angstrom units dataset collected). Datasets (2.6 Angstrom units resolution) of crystals soaked in cyanocobalamin (vitamin B12) have also been collected. BtuB mutants with altered cobalamin binding will be made and characterized, and these mutant structures will be solved. BtuB couples to the inner membrane via the TonB protein. We have expressed a domain of the TonB protein that interacts with BtuB, and will undertake structural studies of this domain alone and in complex with BtuB (also in the presence or absence of vitamin B12). Also, although the natural ligands for BtuB are cobalamins, BtuB is also the receptor to which bacterial toxins (colicins) and bacteriophage bind. The nature of these interactions will be investigated by making, crystallizing and solving complexes of receptor-binding domains of E colicins and BtuB.
