Access Type

Open Access Dissertation

Date of Award

1-1-2010

Degree Type

Dissertation

Degree Name

Ph.D.

Department

Biochemistry and Molecular Biology

First Advisor

Bharati Mitra

Abstract

ZntA from Escherichia coli is a member of the PIBtype ATPase family of transporters. The PIB-type ATPase pumps maintain cellular homeostasis of heavy metals such as Zn2+, Co2+, Cu2+, Cu+, and mediate resistance to toxic metals such as Pb2+, Cd2+ and Ag+. ZntA confers resistance to Pb2+, Cd2+, and Zn2+ by pumping these ions out of the cytoplasm. ZntA has two metal binding sites, one in the hydrophilic N-terminal domain and the other in the transmembrane region. The cysteine-rich N-terminal domain has ~110 amino acids and the conserved motifs -CCCDXXC- and -DCXXC-. The role of these motifs in metal-binding specificity was determined by site-specific mutagenesis, metal-binding studies, functional assays and EXAFS. Our results show that both motifs play a role in metal binding and conferring the broad metal selectivity shown by ZntA.

The N-terminal domain and the N-terminal metal-binding site are not essential for activity, but increase the kinetics of the pump and therefore, confer a survival advantage to the organism. The transmembrane metal-binding site is essential for functioning of the pump. We investigated the role of the N-terminal metal-binding site in the overall function of the pump, and any possible interactions with the transmembrane site. Our results show that the isolated N-terminal domain is capable of transferring its bound metal ion to both wtZntA and to a ZntA mutant lacking the N-terminal domain. Activities obtained with the metal ion bound to the N-terminal domain are much higher than when the metal ion is present as low-affinity complexes in the assay buffer. Together with metal binding affinities of the two sites, these data show that the N-terminal metal site is capable of transferring metal ions directly to the transmembrane metal site. Thus the N-terminal domain acts as a metal chaperone to the transport site in this class of pumps. The function of this "attached" chaperone appears to be to increase the rate of metal binding to the transmembrane site and thus increase the overall kinetics of the pump.

In the last chapter, our studies on a truncated form of ZntA (Del231-ZntA), which lacks the first four transmembrane helices, are presented. This truncated form was based on the natural PINA splice variant of the copper-transporting Wilson disease-associated protein. Del231-ZntA had very low in vitro activity but was able to bind metal ions with a stoichiometry of 0.5, instead of the expected 1.

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