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Access Type

WSU Access

Date of Award

January 2020

Degree Type


Degree Name



Biochemistry and Molecular Biology

First Advisor

Bharati Mitra


ZntA from Escherichia coli belongs to the P1B-ATPase family and mediates resistance to toxic levels of Pb(II), Zn(II), and Cd(II). The N-terminal domain of ZntA is comprised of ~120 residues and contains a single copy of the metal-binding motif GXXCXXD. Additionally, a CPC motif in the fourth transmembrane helix, as well as a conserved Asp714 in the eighth transmembrane helix, form a second metal-binding site in the transmembrane domain of ZntA. A truncated version of the transporter lacking the N-terminal domain, designated ΔN-ZntA, is still able to confer resistance to toxic concentrations of Pb(II), Zn(II), and Cd(II), but functions at a lower catalytic rate. Although the exact role of the N-terminal domain is not established, previous experiments done in our lab show that the hydrophilic N-terminal domain is able to directly transfer metal ions to the transmembrane domain, thus increasing transport activity. For this to occur, a docking interface between the two domains must exist. We hypothesized that this docking takes the form of electrostatic interactions between a region of negatively charged residues in the N-terminal domain and an amphipathic helix in the transmembrane domain containing three positively charged residues: R169, R173, and K176. To investigate this interaction, we first used protein homology modeling software to construct a full model of ZntA and look at potential electrostatic interactions between the two domains. Next, two mutants were constructed at the amphipathic helix: a triple mutant, R169A/R173A/K176A, and a single mutant, R169A. In vivo metal resistance studies and metal-dependent ATPase activity of the triple mutant showed that the overall function of ZntA was affected. The triple mutant resembled ΔN-ZntA with respect to Pb(II) and Zn(II) for the metal resistance studies and ATPase assays, respectively. The R169A mutation did not affect the function of the transporter. This study presents experimental evidence that establishes the amphipathic helix as the docking interface between the N-terminal domain and the transmembrane domain. The next step is examine each of the positive residues singly and doubly in more detail in order to establish the exact residues involved in the docking of the two domains.

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