Nickel Ligation of the N-Terminal Amine of HypA Is Required for Urease Maturation in Helicobacter pylori.
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 six-coordinate site characterized by X-ray absorption spectroscopy (XAS) in unmodified HypA, while another is a diamagnetic four-coordinate planar site characterized by solution nuclear magnetic resonance 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, in which 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 XAS showed a coordination change from six-coordinate in wild-type HypA (WT-HypA) to five-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-HypA compared to those of the Ni sites in 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 that is essential for urease maturation but was at least 20-fold weaker in Ni binding than WT-HypA. 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.
Department of Chemistry and Program of Molecular and Cellular Biology, University of Massachusetts Amherst , Amherst, Massachusetts 01003, United States.
Heidi Q
HQ
Hu
Microbiology and Immunology, Uniformed Services University of the Health Sciences , Bethesda, Maryland 20814, United States.
Ryan C
RC
Johnson
Microbiology and Immunology, Uniformed Services University of the Health Sciences , Bethesda, Maryland 20814, United States.
D Scott
DS
Merrell
Department of Chemistry and Program of Molecular and Cellular Biology, University of Massachusetts Amherst , Amherst, Massachusetts 01003, United States.
Michael J
0000-0002-5598-3038
MJ
Maroney
D000588:Amines / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D016480:Helicobacter pylori / Q000201:enzymology*; D056511:Metallochaperones; D008958:Models, Molecular; D009154:Mutation; D009532:Nickel / Q000378:metabolism*; D020836:Protein Structure, Quaternary; D017433:Protein Structure, Secondary; D014510:Urease / Q000378:metabolism*
D000588:Amines; D001426:Bacterial Proteins; D002352:Carrier Proteins; C578705:HypA protein, Helicobacter pylori; D056511:Metallochaperones; D009532:Nickel; D014510:Urease