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 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 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 microM. 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.

The nickel site of Bacillus pasteurii UreE, a urease metallo-chaperone, as revealed by metal-binding studies and X-ray absorption spectroscopy

STOLA, MASSIMILIANO;MUSIANI, FRANCESCO;ZAMBELLI, BARBARA;CIURLI, STEFANO LUCIANO
2006

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 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 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 microM. 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.
M. Stola; F. Musiani; S. Mangani; P. Turano; N. Safarov; B. Zambelli; S. Ciurli
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11585/32712
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