Structure and dynamics of Helicobacter pylori nickel-chaperone HypA: an integrated approach using NMR spectroscopy, functional assays and computational tools

Abstract

Helicobacter pylori HypA (HpHypA) is a metallochaperone necessary for maturation of [Ni,Fe]-hydrogenase and urease, the enzymes required for colonization and survival of H. pylori in the gastric mucosa. HpHypA contains a structural Zn(II) site and a unique Ni(II) binding site at the N-terminus. X-ray absorption spectra suggested that the Zn(II) coordination depends on pH and on the presence of Ni(II). This study was performed to investigate the structural properties of HpHypA as a function of pH and Ni(II) binding, using NMR spectroscopy combined with DFT and molecular dynamics calculations. The solution structure of apo,Zn-HpHypA, containing Zn(II) but devoid of Ni(II), was determined using 2D, 3D and 4D NMR spectroscopy. The structure suggests that a Ni-binding and a Zn-binding domain, joined through a short linker, could undergo mutual reorientation. This flexibility has no physiological effect on acid viability or urease maturation in H. pylori. Atomistic molecular dynamics simulations suggest that Ni(II) binding is important for the conformational stability of the N-terminal helix. NMR chemical shift perturbation analysis indicates that no structural changes occur in the Zn-binding domain upon addition of Ni(II) in the pH 6.3-7.2 range. The structure of the Ni(II) binding site was probed using ¹H NMR spectroscopy experiments tailored to reveal hyperfine-shifted signals around the paramagnetic metal ion. On this basis, two possible models were derived using quantum-mechanical DFT calculations. The results provide a comprehensive picture of the Ni(II) mode to HpHypA, important to rationalize, at the molecular level, the functional interactions of this chaperone with its protein partners.

Keywords: Computational chemistry; Metal transport; Metallochaperones; Molecular dynamics; Nickel; Nuclear magnetic resonance.

Fig. 1

a ¹H,¹⁵N HSQC spectrum of apo,Zn-HpHypA at 900 MHz at pH 7.2; b ¹H,¹⁵N HSQC spectrum of apo,Zn-HpHypA at 900 MHz at pH 7.2 (blue) overlapped with the spectrum of Ni,Zn-HpHypA (red). The spectra show the relative positions of the peaks, each representing an N-H pair of the amides in the protein main chain. Assignments of individual peaks are shown as blue (for apo,Zn-HpHypA) and red (for Ni,Zn-HpHypA) labels containing the single letter amino acid code and the corresponding residue number. The peak positions are relative to the ¹H and ¹⁵N chemical shift scales of the ω1 and ω2 dimensions, respectively (ppm scales). The pairs of signals corresponding to the amide groups of glutamine/asparagine residues are also shown, with lines linking the HD21/HE21 (left peak) and the HD22/HE22 (right peak) proton signals

Fig. 2

Solution structure of apo,Zn-HpHypA at pH 7.2. a Ribbon diagram of the closest-to-average conformer in the ensemble of the 20 lowest-energy NMR structures, colored from blue in the proximity of the N-terminal to red at the C-terminus; the Zn(II) ion is shown as a blue sphere, coordinated to four thiolate groups of Cys74, Cys 77, Cys91 and Cys94; b ribbon diagram of the structural ensemble of the 20 lowest-energy NMR structures, superimposed on the Ni-binding domain constituted by the three-strand β-sheet and the two α-helices; c “sausage” representation of the NMR ensemble of apo,Zn-HpHypA, colored by secondary structure elements, with the radius proportional to the RMSD of the Cα atoms; d topological diagram of the structure of apo,Zn-HpHypA colored as in a

Fig. 3

Quasi-rigid domain decomposition of apo,Zn-HpHypA trajectory. a SPECTRUS quality score profile. b Domain subdivision of the protein sequence as a function of the number of domains. c Ribbon representation of the most representative structure of the most populated cluster colored according to the optimal domain decomposition into four domains reported in b. d Lys82(Nζ)–Glu71(Cδ) distance variation along simulation time. The gray line represents the effective sampling of the distance during the simulation, while the black line is obtained by applying a Fast Fourier Transform filter to reduce the noise

Fig. 4

The HypA mutant strains are not attenuated for acid survival. The wild-type (WT) strain, urease mutant strain (ΔureB), HypA restorant (ΔhypA-restorant) and HypA mutant strains (ΔhypA::kansacB, G32A, G89A, G32A/G89A, G34A, G104A, and G34A/G104A) were incubated for 1 h in PBS adjusted to pH 6.0 (a, b) or pH 2.3 (c, d), in the absence (a, c) or presence (b, d) of 5 mM urea. Colony forming units (CFU) were enumerated at 0 min (T₀) and 60 min (T₆₀). Two sets of experiments were performed, denoted by the gap and double line in the x-axis, with HypA mutants G32A, G89A, and G32A/G89A tested in the first set, and HypA mutants G34A, G104A, and G34A/G104A tested in the second set. The controls (WT, ΔureB, ΔhypA::kan-sacB, and ΔhypA-restorant) were performed with each set of experiments. Percent survival was calculated as CFU at T₆₀/CFU at T₀ × 100. Data from individual replicates are shown as points, with the bar plotted at the geometric mean. Open symbols indicate that no bacteria were recovered at T₆₀ and are plotted as a function of the limit of detection (100 or 500 CFU/mL for the first and second set of experiments, respectively). Three biological replicates were performed. In a–c, a one-way ANOVA followed by Dunnett’s test for multiple comparisons was performed; the comparisons were made only to WT. In d, the same statistical tests were performed on the log-transformed data. **p < 0.01; ****p < 0.0001

Fig. 5

Chemical shift perturbations (CSP, ppm) obtained by comparing apo,Zn-HpHypA at pH 7.2 and pH 6.3 (H,N in a; Cα,Cβ in b), apo,Zn-HpHypA and Ni,Zn-HpHypA at pH 7.2 (H,N in c; Cα,Cβ in d), apo,Zn-HpHypA and Ni,Zn-HpHypA at pH 6.3 (H,N in e; Cα,Cβ in f), and Ni,Zn-HpHypA at pH 7.2 and pH 6.3 (H,N in g; Cα,Cβ in h). Blue bars indicate the residues comprising the Zn-binding domain

Fig. 6

Comparison of the ¹H,¹⁵N HSQC spectra for Cys74, Cys 77, His79, Cys91, Cys94 and His95 as a function of pH and Ni(II)-binding. Color code: apo,Zn-HpHypA at pH 7.2 = cyan; Ni,Zn-HpHypA at pH 7.2 = blue; apo,Zn-HpHypA at pH 6.3 = orange; Ni,Zn-HpHypA at pH 6.3 = red

Fig. 7

Ribbon diagram of the closest-to-average conformer of the NMR structure ensemble of apo,Zn-HpHypA at pH 7.2, highlighting in red the residues for which the amide NH signals are broadened beyond detection upon binding of paramagnetic Ni(II). The Zn(II) ion is shown as a violet sphere, coordinated by the four thiolate groups of Cys74, Cys 77, Cys91 and Cys94. The side chains of the putative Ni(II)-binding residues Met1, His2, Glu3 and Asp40 are also shown

Fig. 8

400 MHz ¹ NMR spectra of Ni,Zn-HpHypA as a function of pH and presence of deuterated water, at 298 K

Fig. 9

400 MHz ¹H NMR NOE difference spectra of Ni,Zn-HpHypA obtained upon saturation of signals A (red trace), C (blue trace), D (green trace) and H (violet trace); the 1D non-saturated ¹H spectrum is reported in the top trace (black). The signals connected through dipolar interactions are shown

Fig. 10

B3LYP/G 6—311(p,d) optimized geometries of model 1 and 2 for the Ni(II)-binding site in Ni,Zn-HpHypA