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 Ni2+ 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 Ni2+. Hpn-FRET has a moderate selectivity for Ni2+; it also responds to Zn2+ and Co2+ but not to other biometals. Competition experiments between Ni2+ and other metals plus the measured Kd values for Zn2+ and Ni2+ establish the selectivity order for Hpn-FRET as Zn2+ > Ni2+ > Co2+ ≫ 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 Bi3+ to Hpn-FRET was measured to be 6.19 × 10−5 M. Further experiments using Hpn-FRET in E. coli indicate that Hpn-FRET responds to Bi3+ but not to Ni2+ and Zn2+ inside E. coli. The result shows that unlike Ni2+ and Zn2+, which are tightly regulated in most bacteria, available Bi3+ can reach high micromolar levels inside E. coli.