title: The nickel site of Bacillus pasteurii UreE, a urease metallo-chaperone, as revealed by metal-binding studies and X-ray absorption spectroscopy.
abstract: UreE is a homodimeric metallo-chaperone that assists the insertion of Ni(2+) ions in the active site of urease. The crystal structures of UreE from Bacillus pasteurii and Klebsiella aerogenes have been determined, but the details of the nickel-binding site were not elucidated due to solid-state effects that caused disorder in a key portion of the protein. A complementary approach to this problem is described here. Titrations of wild-type Bacillus pasteurii UreE (BpUreE) with Ni(2+), followed by metal ion quantitative analysis using inductively coupled plasma optical emission spectrometry (ICP-OES), established the binding of 2 Ni(2+) ions to the functional dimer, with an overall dissociation constant K(D) = 35 microM. To establish the nature, the number, and the geometry of the ligands around the Ni(2+) ions in BpUreE-Ni(2), X-ray absorption spectroscopy data were collected and analyzed using an approach that combines ab initio extended X-ray absorption fine structure (EXAFS) calculations with a systematic search of several possible coordination geometries, using the Simplex algorithm. This analysis indicated the presence of Ni(2+) ions in octahedral coordination geometry and an average of two histidine residues and four O/N ligands bound to each metal ion. The fit improved significantly with the incorporation, in the model, of a Ni-O-Ni moiety, suggesting the presence of a hydroxide-bridged dinuclear cluster in the Ni-loaded BpUreE. These results were interpreted using two possible models. One model involves the presence of two identical metal sites binding Ni(2+) with negative cooperativity, with each metal ion bound to the conserved His(100) as well as to either His(145) or His(147) from each monomer, residues found largely conserved at the C-terminal. The alternative model comprises the presence of two different binding sites featuring different affinity for Ni(2+). This latter model would involve the presence of a dinuclear metallic core, with one Ni(2+) ion bound to one His(100) from each monomer, and the second Ni(2+) ion bound to a pair of either His(145) or His(147). The arguments in favor of one model as compared to the other are discussed on the basis of the available biochemical data.
authors.affiliation: Laboratory of Bioinorganic Chemistry, Department of Agro-Environmental Science and Technology, University of Bologna, Viale Giuseppe Fanin 40, 40127 Bologna, Italy.
authors.forename: Massimiliano
authors.initials: M
authors.lastname: Stola
authors.forename: Francesco
authors.initials: F
authors.lastname: Musiani
authors.forename: Stefano
authors.initials: S
authors.lastname: Mangani
authors.forename: Paola
authors.initials: P
authors.lastname: Turano
authors.forename: Niyaz
authors.initials: N
authors.lastname: Safarov
authors.forename: Barbara
authors.initials: B
authors.lastname: Zambelli
authors.forename: Stefano
authors.initials: S
authors.lastname: Ciurli
mesh_terms: D015502:Absorptiometry, Photon; D001407:Bacillus; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D001665:Binding Sites; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D018832:Molecular Chaperones / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D009532:Nickel / Q000737:chemistry* / Q000378:metabolism; D014510:Urease / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D002352:Carrier Proteins; D018832:Molecular Chaperones; C090404:UreE protein, Bacteria; D009532:Nickel; D014510:Urease