Nickel Ligation of the N-Terminal Amine of HypA Is Required for Urease Maturation in Helicobacter pylori

Abstract

The human pathogen Helicobacter pylori requires nickel for colonization of the acidic environment of the stomach. HypA, a Ni metallochaperone that is typically associated with hydrogenase maturation, is also required for urease maturation and acid survival of H. pylori. There are two proposed Ni site structures for HypA; one is a paramagnetic 6-coordinate site characterized by X-ray absorption spectroscopy (XAS) in unmodified HypA while another is a diamagnetic four-coordinate planar site characterized by solution NMR in an N-terminally modified HypA construct. To determine the role of the N-terminal amine in Ni binding of HypA, an N-terminal extension variant, L2*-HypA, where a leucine residue was inserted into the second position of the amino acid sequence in the proposed Ni-binding motif, was characterized in vitro and in vivo. Structural characterization of the Ni site using x-ray absorption spectroscopy (XAS) showed a coordination change from 6-coordinate in WT-HypA to 5-coordinate pyramidal in L2*-HypA, which was accompanied by the loss of two N/O-donor protein ligands and the addition of an exogenous bromide ligand from the buffer. The magnetic properties of the Ni-sites in WT- compared to L2*-HypA confirmed that a spin-state change from high- to low-spin accompanied this change in structure. The L2*-HypA H. pylori strain was shown to be acid sensitive and deficient in urease activity in vivo. In vitro characterization showed that L2*-HypA did not disrupt the HypA-UreE interaction essential for urease maturation, but was at least 20-fold weaker in Ni-binding as compared to WT. Characterization of the L2*-HypA variant clearly demonstrates that the N-terminal amine of HypA is involved in proper Ni coordination and is necessary for urease activity and acid survival.

Pathogenesis of Helicobacter pylori infection

“…An optimal balance between nickel uptake and incorporation in metalloenzymes is essential for gastric colonization by H. pylori . Nickel binding is required at the N‐terminal NHE motif of the Ni‐metallochaperone HypA, involved in urease and [NiFe] hydrogenase maturation, which are essential for acid survival of H. pylori . Fischer et al .…”

“…Nickel binding is required at the N-terminal NHE motif of the Ni-metallochaperone HypA, involved in urease and [NiFe] hydrogenase maturation, which are essential for acid survival of H. pylori. 8 Fischer et al 9 identified a novel H. pylori nickel transport system NiuBDE. NiuBDE is required for nickel-dependent urease activation and acid survival.…”

Nickel Metalloregulators and Chaperones

“…The HypA structures reveal that the nickel binding site and the zinc binding site are separated by a long flexible linker (Figures 8 and 9) [98][99][100]. The N-terminal nickel site features a highly conserved MHE motif, which includes the first three amino acids at the N-terminus of the protein, that is used to bind two Ni(II) ions per dimer with micromolar affinity [93][94][95][96]98,101]. A K d of 75 ± 46 nM was reported by Douglas et al for a Strep-tagged HypA protein binding to nickel in competition with a well-established metal binding indicator, Mag-fura 2 [102].…”

“…HypA utilizes two conserved CXXC motifs to bind one zinc ion per monomer [93,94,[97][98][99][100][101] with nanomolar affinity [94]. HypA proteins have been reported as being a dimer in solution based on elution volumes [94,95], however, size exclusion chromatography in combination with multiple light scattering (SEC-MALS) determined that the protein is a monomer in solution [96]. HypA proteins feature two metal binding sites, a N-terminal nickel site and a zinc site [94,95,[97][98][99][100].…”

“…HypA proteins feature two metal binding sites, a N-terminal nickel site and a zinc site [94,95,[97][98][99][100]. The HypA structures reveal that the nickel binding site and the zinc binding site are separated by a long flexible linker (Figures 8 and 9 H. pylori HypA binds Ni(II) with a Kd of ~1 μM at pH 7.2 and this binding is weaker at pH 6.3 [96,97,103]. There are some discrepancies regarding the coordination number and geometry of the nickel biding site determined by XAS and X-ray crystallography.…”