The structure of the high-affinity nickel-binding site in the Ni,Zn-HypA•UreE2 complex

Autor: Barbara Zambelli, Priyanka Basak, Heidi Hu, Mario Piccioli, Francesco Musiani, Valquiria Broll, Lionel Imbert, Jerome Boisbouvier, Michael J Maroney, Stefano Ciurli
Přispěvatelé: Alma Mater Studiorum Università di Bologna [Bologna] (UNIBO), University of Massachusetts [Amherst] (UMass Amherst), University of Massachusetts System (UMASS), CERM and Deparment of Chemistry, Università degli Studi di Firenze = University of Florence (UniFI), University of Bologna/Università di Bologna, Institut de biologie structurale (IBS - UMR 5075), Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes (UGA), Department of Agro-Environmental Science and Technology
Rok vydání: 2023
Předmět:
Zdroj: Metallomics
Metallomics, 2023, 15 (3), ⟨10.1093/mtomcs/mfad003⟩
ISSN: 1756-591X
1756-5901
DOI: 10.1093/mtomcs/mfad003
Popis: 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.
Databáze: OpenAIRE
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