The <i>Helicobacter pylori</i> HypA·UreE₂ Complex Contains a Novel High-Affinity Ni(II)-Binding Site

<i>Helicobacter pylori</i> is a human pathogen that colonizes the stomach, is the major cause of ulcers, and has been associated with stomach cancers. To survive in the acidic environment of the stomach, <i>H. pylori</i> uses urease, a nickel-dependent enzyme, to produce ammonia for maintenance of cellular pH. The bacteria produce apo-urease in large quantities and activate it by incorporating nickel under acid shock conditions. Urease nickel incorporation requires the urease-specific metallochaperone UreE and the (UreFGH)₂ maturation complex. In addition, the <i>H. pylori</i> nickel urease maturation pathway recruits accessory proteins from the [NiFe] hydrogenase maturation pathway, namely, HypA and HypB. HypA and UreE dimers (UreE₂) are known to form a protein complex, the role of which in urease maturation is largely unknown. Herein, we examine the nickel-binding properties and protein–protein interactions of HypA and UreE₂ using isothermal titration calorimetry and fluorometric methods under neutral and acidic pH conditions to gain insight into the roles played by HypA in urease maturation. The results reveal that HypA and UreE₂ form a stable complex with micromolar affinity that protects UreE from hydrolytic degradation. The HypA·UreE₂ complex contains a unique high-affinity (nanomolar) Ni²⁺-binding site that is maintained under conditions designed to mimic acid shock (pH 6.3). The data are interpreted in terms of a proposed mechanism wherein HypA and UreE₂ act as co-metallochaperones that target the delivery of Ni²⁺ to apo-urease with high fidelity