UreE-UreG complex facilitates nickel transfer and preactivates GTPase of UreG in Helicobacter pylori

Abstract

The pathogenicity of Helicobacter pylori relies heavily on urease, which converts urea to ammonia to neutralize the stomach acid. Incorporation of Ni(2+) into the active site of urease requires a battery of chaperones. Both metallochaperones UreE and UreG play important roles in the urease activation. In this study, we demonstrate that, in the presence of GTP and Mg(2+), UreG binds Ni(2+) with an affinity (Kd) of ∼0.36 μm. The GTPase activity of Ni(2+)-UreG is stimulated by both K(+) (or NH4 (+)) and HCO3 (-) to a biologically relevant level, suggesting that K(+)/NH4 (+) and HCO3 (-) might serve as GTPase elements of UreG. We show that complexation of UreE and UreG results in two protein complexes, i.e. 2E-2G and 2E-G, with the former being formed only in the presence of both GTP and Mg(2+). Mutagenesis studies reveal that Arg-101 on UreE and Cys-66 on UreG are critical for stabilization of 2E-2G complex. Combined biophysical and bioassay studies show that the formation of 2E-2G complex not only facilitates nickel transfer from UreE to UreG, but also enhances the binding of GTP. This suggests that UreE might also serve as a structural scaffold for recruitment of GTP to UreG. Importantly, we demonstrate for the first time that UreE serves as a bridge to grasp Ni(2+) from HypA, subsequently donating it to UreG. The study expands our horizons on the molecular details of nickel translocation among metallochaperones UreE, UreG, and HypA, which further extends our knowledge on the urease maturation process.

Keywords: Helicobacter pylori; Metal Ion-Protein Interaction; Metallochaperone; Metalloprotein; Nickel; Protein-Protein Interaction; Translocation; UreE; UreG; Urease; Urease Maturation.

FIGURE 1.

Ni²⁺ binding to UreG monitored by UV-visible spectroscopy. A–D, Ni²⁺ was titrated into 20 μm apo-UreG in 20 mm Hepes, 100 mm NaCl, 500 μm TCEP, pH 7.2, with supplementation of 100 μm GTP, 1 mm Mg²⁺ (A), 100 μm GDP, 1 mm Mg²⁺ (B), only 100 μm GTP without Mg²⁺ (C), or 20 μm Zn²⁺, 100 μm GTP, and 1 mm Mg²⁺ (D). The titration curve plotted at 337 nm is shown in the inset. Abs, absorbance.

FIGURE 2.

GTPase activity of UreG and its effect on urease activation. A, GTPase activity of Ni-UreG in the presence of K⁺ (KCl), HCO₃⁻ (NaHCO₃), or KHCO₃ with concentrations ranging from 0 to 100 mm. (GTPase activity is defined as nmol/min/mg of protein.) B, GTPase activity of Ni-UreG at different pH values in the presence of 10 mm K⁺, HCO₃⁻, or KHCO₃. C, GTPase activity of apo-UreG at different pH values. D, GTPase activity of Zn-UreG at different pH values. The GTPase activity of Ni-UreG is shown as a dotted line for comparison. All GTPase assay buffer contains 1 mm MgSO₄. E, effect of Ni-UreG on urease activation. Note that significant enhanced activity of urease was found only in the present of both Ni-UreG and KHCO₃. (The unit of urease activity is defined as nmol of ammonia produced per min/mg of total protein.) Error bars indicate means ± S.E.

FIGURE 3.

Formation of UreE-UreG complex. UreE and UreG (40 μm for each) samples were incubated with 1.5 molar eq of metal ions (Ni²⁺/Zn²⁺/Mg²⁺) with or without supplementation of guanine nucleotides (GDP or GTP) and loaded onto an analytic column. A, the formation of UreE-UreG complex in the absence of Mg²⁺. Profiles of free UreE and UreG are shown for comparison (lines 1 and 2). B, the formation of UreE-UreG complex in the presence of Mg²⁺. Note that 2E-2G complex is formed only in the presence of both GTP and Mg²⁺ (lines 5, 7, and 9).

FIGURE 4.

Examination of UreE and UreG mutants on the protein-protein interaction by chromatography. UreE and UreG or their variants (40 μm for each) were incubated in a buffer containing 60 μm Mg²⁺ in the absence (black curves) or presence (colored curves) of 60 μm GTP. Note that the single mutation R101A on UreE (line 2) and C66A on UreG (line 6) partially or completely disrupts the formation of 2E-2G complex, whereas the excess Ni²⁺ restores the formation of 2E-2G complex for the UreE-R101A-UreG mixture (line 2, broken line).

FIGURE 5.

Ni²⁺ translocation between UreE and UreG monitored by UV-visible spectroscopy and chromatography. A and B, UV-visible spectra of 25 μm UreG (A) and Ni-UreG (B) titrated stepwise with Ni²⁺-UreE, apo-UreE in Hepes buffer, pH 7.2, containing 50 μm GTP and 1 mm Mg²⁺. The appearance of peaks at 337 nm in A indicates that Ni²⁺ is transferred from UreE to UreG. Abs, absorbance. C, UreG (25 μm) was titrated with Ni²⁺-CUreE in the presence of GTP (50 μm) and Mg²⁺ (1 mm). D, UreG (25 μm) was titrated with Ni²⁺-UreE-R101A in the presence of GTP (50 μm) and Mg²⁺ (1 mm). E, UreG (25 μm) was titrated with Ni²⁺-UreE in the presence of GTP (50 μm) and absence of Mg²⁺. The weak peak at 337 nm (D and E) indicates the inefficient Ni²⁺ transfer via 2E-G complex. The addition of Mg²⁺ (100 μm) raises the peak intensities at 337 nm (E, broken line), implying that Ni²⁺ transfer from UreE to UreG is restored upon the formation of 2E-2G complex. F, gel filtration profiles of UreE-UreG mixtures. UreG was incubated with a series of UreE proteins loaded with different molar equivalents of Ni²⁺ (0, 0.3, 0.6, and 1.0) prior to the injection. The binding of Ni²⁺ induces dimerization of UreG.

FIGURE 6.

Enhancement of GTPase activity of UreG by UreE. A, time dependence of GTPase activities of apo-UreG in the presence of 1 molar eq of UreE loaded with different amounts of Ni²⁺ (0-, 0.3-, 0.6-, and 1.0-fold). The black curve represents the GTPase activity of Ni-UreG. B, time dependence of GTPase activities of Ni-UreG in the presence of different amounts of apo-UreE (0-, 0.3-, 0.6-, and 1.0-fold). C, saturation curves of Ni-UreG in the absence and presence of equimolar amounts of apo-UreE. K_m values are obtained by fitting the saturation curves to the Michaelis-Menten equation to be ∼82.7 × 10⁻⁶ m for Ni-UreG alone and 27.9 × 10⁻⁶ m for Ni-UreG and UreE mixture. GTPase assays were carried out in the presence of 10 mm KHCO₃. GTP conc., GTP concentration. Error bars indicate means ± S.E. D, gel filtration chromatography profiles of UreG (∼40 μm) preincubated with different amounts of UreE (0, 20, 40, 60 μm) supplemented with ∼40 μm GTP and 1 mm MgSO₄. The addition of UreE into UreG leads to the formation of 2E-2G complex, facilitating GTP binding.

FIGURE 7.

Complexation of UreE, UreG, and HypA and Ni²⁺ translocation. A, analytic gel filtration profiles of HypA, UreE, and UreG in the absence (blue curve) or presence (orange curve) of GTP and Mg²⁺. mAU, milliabsorbance units. B, effect of Ni-HypA on the GTPase activity of UreG. Note that in the presence of UreE, Ni-HypA enhances the activity of UreG to a level similar to that of the Ni-UreE and UreG mixture. GTPase assays were carried out in the presence of 10 mm KHCO₃. Error bars indicate means ± S.E. C, UreG (20 μm) was titrated with Ni-Zn-HypA in the presence of GTP (50 μm) and Mg²⁺ (1 mm). Abs, absorbance. D, UreG (20 μm) was titrated with a mixture of Ni-Zn-HypA and UreE in the presence of GTP (50 μm) and Mg²⁺ (1 mm).

FIGURE 8.

Proposed mechanism of Ni²⁺ translocation among HypA, UreE, and UreG during urease maturation. Apo-UreE acquires Ni²⁺ from HypA via specific HypA-UreE interaction. Sequentially, Ni-UreE dimer binds one apo-UreG monomer and then further captures the second UreG monomer, facilitating GTP binding to UreG in the presence of Mg²⁺, or Ni-UreE dimer binds two apo-UreG monomers in the presence of GTP and Mg²⁺ to form 2E-2G complex, which triggers nickel translocation from UreE to UreG. Subsequently, UreF-H competes with UreE for Ni-UreG to form the supercomplex as an apo-urease/UreF-H-G (36), in which the GTP hydrolysis by UreG is catalyzed to complete the final step of nickel insertion into the apo-urease in the presence of KHCO₃/NH₄HCO₃.