Structure-function analyses of metal-binding sites of HypA reveal residues important for hydrogenase maturation in Helicobacter pylori

Abstract

The nickel-containing enzymes of Helicobacter pylori, urease and hydrogenase, are essential for efficient colonization in the human stomach. The insertion of nickel into urease and hydrogenase is mediated by the accessory protein HypA. HypA contains an N-terminal nickel-binding site and a dynamic structural zinc-binding site. The coordination of nickel and zinc within HypA is known to be critical for urease maturation and activity. Herein, we test the hydrogenase activity of a panel of H. pylori mutant strains containing point mutations within the nickel- and zinc-binding sites. We found that the residues that are important for hydrogenase activity are those that were similarly vital for urease activity. Thus, the zinc and metal coordination sites of HypA play similar roles in urease and hydrogenase maturation. In other pathogenic bacteria, deletion of hydrogenase leads to a loss in acid resistance. Thus, the acid resistance of two strains of H. pylori containing a hydrogenase deletion was also tested. These mutant strains demonstrated wild-type levels of acid resistance, suggesting that in H. pylori, hydrogenase does not play a role in acid resistance.

Fig 1. The structure of HypA and its role in urease and hydrogenase maturation.

(A) Representation of the NMR structure of H. pylori HypA (PDB: 2KDX) [25] with the main chain colored in light grey and the metal binding sites in color to highlight the location of residues involved in metal coordination. Residues comprising the nickel-binding site (M1, H2, E3, and D40) are shown in green. Residues of the zinc-binding site (C74, C77, H79, C91, C94, and H95) are shown in cyan. The metal-binding oxygen (red), nitrogen (blue), and sulfur (yellow) atoms are shown as small spheres. The nickel atom representation in this figure (dotted green circle) was not resolved in the 2KDX structure, and the resolved zinc atom is shown as a dark grey sphere. The zinc-binding site adopts two pH-dependent conformations, as illustrated: Zn(Cys)₂(His)₂ at acidic pH, and Zn(Cys)₄ at neutral pH. (B) HypA contributes to the maturation of hydrogenase and urease through delivery of nickel (green circles). Urease requires nickel for activity, of which one of the downstream effects is acid resistance. In the absence of HypA, maturation of urease can still be accomplished through the addition of excess nickel (dashed line). Hydrogenase requires nickel for activity, but herein is shown not to contribute to in vitro acid resistance (red X). In the absence of HypA, maturation of hydrogenase cannot be accomplished through the addition of excess nickel.

Fig 2. H. pylori ΔhydB has no detectable hydrogenase activity and wild-type urease activity.

(A) Cell lysates of the wild-type (WT) strain, urease mutant strain (ΔureB), and hydrogenase mutant strain (ΔhydB) were used to measure hydrogenase activity using a methyl viologen assay. The rate at which H₂ was oxidized (in μmol/min) was obtained using the slope of absorbance at A_{578 nm}, which was normalized to the amount of total protein in the cell lysate (in μg), and normalized against the activity of the WT strain to obtain percent hydrogenase activity. Three biological replicates were tested for each strain, and the mean and standard deviation are graphed. (B) Cell lysates of the WT, ΔureB, and ΔhydB strains were used to measure urease activity using ammonia production in a phenol-hypochlorite assay. The specific urease activity was normalized to the amount of total protein in the cell lysate (in μg), and normalized against the specific activity of the WT strain to obtain relative urease activity. Three biological replicates were tested for each strain, and the mean and standard deviation are graphed.

Fig 3. The ΔhydB strain of H. pylori G27 is not attenuated for acid survival.

The wild-type (WT) strain, urease mutant strain (ΔureB), and hydrogenase mutant strain (ΔhydB) were incubated for 1 hr in PBS adjusted to pH 6.0 (A and B) or to pH 2.3 (C and D), in the absence (A and C) or presence (B and D) of 5 mM urea. The number of colony-forming units (CFU) was measured at 0 min (T₀) and at 60 min (T₆₀), and percent survival was calculated as CFU at T₆₀ divided by CFU at T₀. Data from individual biological replicates are shown as points, with the bar plotted at the mean. Open symbols indicate that no bacteria were recovered and thus, the CFU are plotted as a function of the limit of detection (100 CFU/mL). Three biological replicates were performed. For panels A-C, a one-way ANOVA followed by Dunnett’s test for multiple comparisons was performed; the comparison was made only to WT. In panel D, the same statistical tests were performed on the log-transformed data. **** = p < 0.0001.

Fig 4. The ΔhydABCDE strain of H. pylori 26695 is not attenuated for acid survival.

The wild-type (WT) strain, urease mutant strain (ΔureAB), and hydrogenase mutant strain (ΔhydABCDE) were incubated for 1 hr in PBS adjusted to pH 6.0 (A and B) or to pH 2.3 (C and D), in the absence (A and C) or presence (B and D) of 5 mM urea. The number of colony-forming units (CFU) was measured at 0 min (T₀) and at 60 min (T₆₀), and percent survival was calculated as CFU at T₆₀ divided by CFU at T₀. Data from individual biological replicates are shown as points, with the bar plotted at the mean. Open symbols indicate that no bacteria were recovered and thus, the CFU are plotted as a function of the limit of detection (1000 CFU/mL). Three biological replicates were performed. For panels A-C, a one-way ANOVA followed by Dunnett’s test for multiple comparisons was performed; the comparison was made only to WT. In panel D, the same statistical tests were performed on the log-transformed data. **** = p < 0.0001.

Fig 5. Mutation of the metal coordination sites of HypA results in decreased hydrogenase activity.

Cell lysates from the indicated hypA mutant strains, in addition to wild-type (WT) strain, urease mutant strain (ΔureB), hypA mutant strain (hypA::kan-sacB), and hypA restorant (hypA-R) were utilized to determine hydrogenase activity using a methyl viologen assay. The rate at which H₂ was oxidized (in μmol/min) was obtained using the slope of absorbance at A_{578 nm}, which was normalized to the amount of total protein in the cell lysate (in μg), and normalized against the activity of the WT strain to obtain percent hydrogenase activity. The hydrogenase activities of hypA mutant strains with mutations found within the nickel-binding site (A) and within the zinc-binding site (B) are shown. Two biological replicates were tested in A, and three biological replicates were tested in B. The mean is graphed, with range (A) or standard deviation (B).