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Author ORCID Identifier



Open Access Dissertation

Document Type


Degree Name

Doctor of Philosophy (PhD)

Degree Program

Molecular and Cellular Biology

Year Degree Awarded


Month Degree Awarded


First Advisor

Michael J. Maroney

Second Advisor

Michael J. Knapp

Third Advisor

Jeanne A. Hardy

Fourth Advisor

Peter Chien

Subject Categories

Biochemistry | Biophysics | Molecular Biology | Other Biochemistry, Biophysics, and Structural Biology | Pathogenic Microbiology | Structural Biology


Helicobacter pylori is a bacterium that has colonized the human gastric mucosa of over 50% of the world population. Persistent infection can cause gastritis, peptic ulcers, and cancers. The ability of H. pylori to colonize the acidic environment of the human stomach is dependent on the activity of the nickel containing enzymes, urease and NiFe-hydrogenase. The nickel metallochaperone, HypA, was previously shown to be required for the full activity of both enzymes. In addition to a Ni-binding site, HypA also contains a structural Zn site, which has been characterized to alter its averaged structure depending on pH and the presence of nickel.

Site-directed mutagenesis of the HypA Zn- and Ni-binding residues were used to elucidate mechanistic details in the involvement of HypA in Ni-delivery to enzymatic targets. Cellular studies were conducted to characterize the acid survival and enzymatic activities in a panel of hypA isogenic strains of H. pylori in collaboration with geneticists at the Uniformed Services University of Health Sciences (Bethesda, MD). In vitro studies were conducted on purified HypA variant proteins, correlating structural changes to the metal binding sites, and further probing the biophysical interactions with Ni(II) and protein binding partners.

Our studies indicated that NiFe-hydrogenase was not required for acid survival in H. pylori cultures. Only mutations of the conserved Cys residues in the HypA Zn site were found to exhibit varying degrees of acid sensitivity and loss of activity in Ni-containing enzymes. The same Cys residues were found to be important for the maturation of both urease and NiFe-hydrogenase, indicating that these residues were critical for the function of HypA as a metallochaperone. Our mutagenesis studies found that Ni(II) binds to HypA at the N-terminus, coordinating the N-terminal amine of Met1 as an essential ligand, the loss of which lead to acid sensitivity and loss of enzymatic activities in isogenic strain. We also found that protein-protein interactions between HypA and dimeric UreE (an essential Ni-metallochaperone for urease) were enhanced in the presence of Ni(II) in both neutral and acidic conditions. Further, the presence of HypA protected UreE from degradation. Taken together, we concluded that HypA is acting as a co-metallochaperone along with dimeric UreE in Ni-delivery for urease maturation.