Unraveling the Helicobacter pylori UreG zinc binding site using X-ray absorption spectroscopy (XAS) and structural modeling

Abstract

The pathogenicity of Helicobacter pylori depends on the activity of urease for pH modification. Urease activity requires assembly of a dinickel active site that is facilitated in part by GTP hydrolysis by UreG. The proper functioning of Helicobacter pylori UreG (HpUreG) is dependent on Zn(II) binding and dimerization. X-ray absorption spectroscopy and structural modeling were used to elucidate the structure of the Zn(II) site in HpUreG. These studies independently indicated a site at the dimer interface that has trigonal bipyramidal geometry and is composed of two axial cysteines at 2.29(2)Å, two equatorial histidines at 1.99(1)Å, and a solvent-accessible coordination site. The final model for the Zn(II) site structure was determined by refining multiple-scattering extended X-ray absorption fine structure fits using the geometry predicted by homology modeling and ab initio calculations.

UreE-UreG Complex Facilitates Nickel Transfer and Preactivates GTPase of UreG in Helicobacter pylori

“…It has been shown that both Ni 2ϩ and Zn 2ϩ bind UreG through coordination of Cys-66 and His-68 (40). Although the binding affinity of UreG to Ni 2ϩ determined in the present , implying that UreG is a specific nickel binding chaperone.…”

“…A previous x-ray absorption spectroscopy study showed that the coordination sphere for zinc is formed by residues Cys-66 and His-68 from each of the monomers of HpUreG dimer (40 (36), whereas the HpUreG counterpart, HpHypB, shows a higher GTPase activity in solution containing potassium (K ϩ ) (24), implying the potential roles of K ϩ and HCO 3 Ϫ in GTP hydrolysis. To verify this hypothesis, GTPase assay was carried out in a buffer containing 1 mM MgSO 4 supplemented with KCl, NaHCO 3 , KHCO 3 , or NH 4 HCO 3 at concentrations of 0, 0.5, 1, 5, 10, 20, 30, 40, 50, 100 mM, and the pH of the buffer was adjusted carefully to be 7.5.…”

An XAS investigation of the nickel site structure in the transcriptional regulator InrS

“…X-ray absorption spectroscopy (XAS) data were collected as previously described[8, 9] on beam line X3B at the National Synchrotron Light Source (NSLS), Brookhaven National Laboratories (Upton, NY), except for Ni(II)-H21L, which used beam line 7-3 at the Stanford Synchrotron Radiation Laboratory (SSRL). Data collected at NSLS used samples that were cooled to ~30 K using a He displex cryostat and were collected under ring conditions of 2.8 GeV and 120–300 mA, with a sagittally-focusing Si(111) double-crystal monochromator.…”

“…The distances of the five non-hydrogen atoms in the imidazole ring were thus fit in terms of a single M-N distance for various angles of rotation (α = 0 – 10°)[13–15]. Multiple-scattering parameters for imidazole ligands bound to Ni(II) and Cu(II) were generated using the FEFF6 software package with the imidazole input obtained from average bond lengths and angles gathered from crystallographic data, as previously described[8, 16]. Histidine “counting” was performed by adding integer numbers of His imidazole ligands to fits and assessing the effect on the R- factor and reduced χ 2 ( vide infra ).…”

Biosynthesis of the Urease Metallocenter

“…X-ray absorption spectroscopy of the zincbound protein revealed a trigonal bipyramidal site including two His and two Cys residues, likely positioned at the subunit interface (49). The soybean (Glycine max) protein also exhibits a monomer/dimer equilibrium, with the dimer stabilized by Zn 2ϩ , but the binding thermodynamics are quite complex (47).…”