Characterization of a nucleotide binding domain associated with Neisserial iron transport

The ability for the pathogen Neisseria gonorrhoeae to sequester iron from sources such as transferrin from the human host plays an important role in initiating infection. The Neisseria! fbpABC operon encodes an ABC (ATP binding cassette) transporter proposed to function in transporting iron at the periplasm-to-cytosol level. The highly conserved ATP binding domain of these transporters typically utilizes the energy of ATP hydrolysis to pump substrates across the membrane against a concentration gradient. The goal of my project is to show that FbpC functions as a nucleotide binding domain for this iron transport system. First, the N. gonorrhoeae fbpC gene was successfully amplified and cloned into the pET28a expression vector. The resulting fusion protein (FbpC(his6)) of approximately 40kDa was overexpressed in Escherichia coli HMS174(DE3) cells and purified to near homogeneity by nickel-chelate affinity followed by anion-exchange chromatography. Isolated FbpC(his6) has intrinsic ATPase activity uncoupled from the iron transport process and displays a specific activity of approximately 0.5 μmol/min/mg, similar to that determined for the distantly related nucleotide binding domains HisP and MalK in their purified forms. An FbpC mutant, E164D, designed to be defective in ATP hydrolysis was produced, purified, and found to contain a ten-fold reduction in specific activity as compared to the wild-type. Purified FbpC(his6) was also covalently modified by 8-azido-[γ32P]ATP, and this interaction was shown to be specific by preincubation of reactions with unlabeled ATP. In conclusion, FbpC is a functional nucleotide binding domain capable of powering the iron transporter. [Scientific formulae used in this abstract could not be reproduced.]Medicine, Faculty ofBiochemistry and Molecular Biology, Department ofGraduat