The maturation pathway for the nickel-dependent enzyme urease utilizes the protein UreE as a metallochaperone to supply Ni(II) ions. In Helicobacter pylori urease maturation also requires HypA and HypB, accessory proteins that are commonly associated with hydrogenase maturation. Herein we report on the characterization of a protein complex formed between HypA and the UreE2 dimer. Nuclear magnetic resonance (NMR) coupled with molecular modelling show that the protein complex apo, Zn-HypA•UreE2, forms between the rigorously conserved Met-His-Glu (MHE motif) Ni-binding N-terminal sequence of HypA and the two conserved His102A and His102B located at the dimer interface of UreE2. This complex forms in the absence of Ni(II) and is supported by extensive protein contacts that include the use of the C-terminal sequences of UreE2 to form additional strands of β-sheet with the Ni-binding domain of HypA. The Ni-binding properties of apo, Zn-HypA•UreE2 and the component proteins were investigated by isothermal titration calorimetry using a global fitting strategy that included all of the relevant equilibria, and show that the Ni,Zn-HypA•UreE2 complex contains a single Ni(II)-binding site with a sub-nanomolar KD. The structural features of this novel Ni(II) site were elucidated using proteins produced with specifically deuterated amino acids, protein point mutations, and the analyses of X-ray absorption spectroscopy, hyperfine shifted NMR features, as well as molecular modeling coupled with quantum-mechanical calculations. The results show that the complex contains a six-coordinate, high-spin Ni(II) site with ligands provided by both component proteins.
Keywords: Helicobacter pylori; enzyme maturation; metallochaperone; nickel trafficking.
© The Author(s) 2023. Published by Oxford University Press.