The Nickel Site of Bacillus pasteurii UreE, a Urease Metallo-Chaperone, As Revealed by Metal-Binding Studies and X-ray Absorption Spectroscopy

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Abstract
UreE is a homodimeric metallo-chaperone that assists the insertion of Ni2+ ions in the active site of urease. The crystal structures of UreE from Bacilluspasteurii and Klebsiellaaerogenes 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 Bacilluspasteurii UreE (BpUreE) with Ni2+, followed by metal ion quantitative analysis using inductively coupled plasma optical emission spectrometry (ICP-OES), established the binding of 2 Ni2+ ions to the functional dimer, with an overall dissociation constant KD = 35 μM. To establish the nature, the number, and the geometry of the ligands around the Ni2+ ions in BpUreE-Ni2, 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 Ni2+ 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 Ni2+ with negative cooperativity, with each metal ion bound to the conserved His100 as well as to either His145 or His147 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 Ni2+. This latter model would involve the presence of a dinuclear metallic core, with one Ni2+ ion bound to one His100 from each monomer, and the second Ni2+ ion bound to a pair of either His145 or His147. The arguments in favor of one model as compared to the other are discussed on the basis of the available biochemical data.