Nickel binding shifts Helicobacter pylori HypA toward compact conformations

Helicobacter pylori (Hp) is a Gram-negative human pathogen that relies on the nickel enzymes urease and [Ni, Fe]-hydrogenase for gastric colonization. Delivery of Ni(II) to the active sites of these enzymes is mediated by the metallochaperone HpHypA, which contains a high-affinity structural Zn(II) site and a lower-affinity Ni(II) site, in two distinct domains. Although the NMR solution structure of the apo form Zn-HpHypA is known, the impact of Ni(II) binding on the structure remains unclear. Here, NMR 15 N relaxation experiments have been applied to probe the backbone internal dynamics and the rotational diffusion of Zn-HpHypA and Ni,Zn-HpHypA. Residueresolved model-free analysis revealed broadly similar ps-ns backbone motional amplitudes in the apo and holo states. In contrast, Ni(II) binding increased rotational diffusion, indicating a reduced hydrodynamic radius and a shift toward a more compact conformational ensemble. These observations support a model in which Ni(II) binding generates a delivery-competent protein structure by narrowing the accessible conformational distribution during partner recognition. Previously, calorimetric data reported on the interaction between HpHypA and the urease metallochaperone homodimeric HpUreE 2 , which showed micromolar Ni(II) binding to HpHypA but the emergence of a sub-nanomolar, strongly exothermic Ni site in the HypA center dot UreE 2 complex. The observed nickelinduced ensemble compaction determined by solution NMR spectroscopy provides a plausible physical basis for Ni-dependent conformational switching to promote high-affinity protein-protein recognition.