Metal-binding properties of Hpn from Helicobacter pylori and implications for the therapeutic activity of bismuth

Nickel is of particular importance to Helicobacter pylori in part because it acts as a cofactor of urease, which is critical to the survival of H. pylori. In this study the nickel storage, histidine-rich protein Hpn from H. pylori was converted into a Ni super(2+) probe by inserting it between two fluorescence resonance energy transfer (FRET) partners, cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP). The resulting construct, Hpn-FRET, exhibited a change in FRET upon the binding of Ni super(2+). Hpn-FRET has a moderate selectivity for Ni super(2+); it also responds to Zn super(2+) and Co super(2+) but not to other biometals. Competition experiments between Ni super(2+) and other metals plus the measured K sub(d) values for Zn super(2+) and Ni super(2+) establish the selectivity order for Hpn-FRET as Zn super(2+) > Ni super(2+) > Co super(2+) >> other biometals. Bismuth is widely used as a therapeutic agent against H. pylori, and Hpn has been suggested as one of the possible targets. The dissociation constant of Bi super(3+) to Hpn-FRET was measured to be 6.19 x 10 super(-5) M. Further experiments using Hpn-FRET in E. coli indicate that Hpn-FRET responds to Bi super(3+) but not to Ni super(2+) and Zn super(2+) inside E. coli. The result shows that unlike Ni super(2+) and Zn super(2+), which are tightly regulated in most bacteria, available Bi super(3+) can reach high micromolar levels inside E. coli.