title: Nickel translocation between metallochaperones HypA and UreE in Helicobacter pylori.
abstract: Incorporation of nickel ions to the active sites of urease and hydrogenase is prerequisite for the appropriate functions of the metalloenzymes. Such a process requires the participation of several accessory proteins. Interestingly, some of them are shared by the two enzymes in their maturation processes. In this work, we characterized the molecular details of the interaction of metallochaperones UreE and HypA in Helicobacter pylori. We show by chemical cross-linking and static light scattering that the UreE dimer binds to HypA to form a hetero-complex i.e. HypA-(UreE)2. The dissociation constant (Kd) of the protein complex was determined by ITC to be 1 μM in the absence of nickel ions; whereas binding of Ni(2+) but not Zn(2+) to UreE resulted in ca. one fold decrease in the affinity. The putative interfaces on HypA unveiled by NMR chemical shift perturbation were found mainly at the nickel binding domain and in the cleft between α1 and β1/β6. We also identified that the C-domain of UreE, in particular the C-terminal residues of 158-170 are indispensable for the interaction of UreE and HypA. Such an interaction was also observed intracellularly by GFP-fragment reassembly assay. Moreover, we demonstrated using a fluorescent probe that nickel is transferred from HypA to UreE via the specific protein-protein interaction. Deletion of the C-terminus (residues 158-170) of UreE abolished nickel transfer and led to a significant decrease in urease activity. This study provides direct in vitro and in vivo evidence as well as molecular details of nickel translocation mediated by protein-protein interaction.
authors.affiliation: Department of Chemistry, The University of Hong Kong, Pokfulam Road, Hong Kong, P.R. China. hsun@hku.hk.
authors.forename: Xinming
authors.initials: X
authors.lastname: Yang
authors.forename: Hongyan
authors.initials: H
authors.lastname: Li
authors.forename: Tianfan
authors.initials: T
authors.lastname: Cheng
authors.forename: Wei
authors.initials: W
authors.lastname: Xia
authors.forename: Yau-Tsz
authors.initials: YT
authors.lastname: Lai
authors.forename: Hongzhe
authors.initials: H
authors.lastname: Sun
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D002151:Calorimetry; D002352:Carrier Proteins / Q000737:chemistry / Q000378:metabolism*; D057075:Enzyme Assays; D004926:Escherichia coli / Q000378:metabolism; D005456:Fluorescent Dyes / Q000378:metabolism; D016480:Helicobacter pylori / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy; D056511:Metallochaperones / Q000378:metabolism*; D009532:Nickel / Q000378:metabolism*; D011485:Protein Binding; D014510:Urease / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D002352:Carrier Proteins; D005456:Fluorescent Dyes; C578705:HypA protein, Helicobacter pylori; D056511:Metallochaperones; C090404:UreE protein, Bacteria; D009532:Nickel; D014510:Urease