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

Abstract

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 K_d 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.