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Free Content Chimeras of P1-type ATPases and their transcriptional regulators: contributions of a cytosolic amino-terminal domain to metal specificity

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Zn2+-responsive repressor ZiaR and Co2+-responsive activator CoaR modulate production of P1-type Zn2+- (ZiaA) and Co2+- (CoaT) ATPases respectively. What dictates metal selectivity? We show that Δ¬†ziaΔcoa double mutants had similar Zn2+ resistance to Δzia single mutants and similar Co2+ resistance to Δcoa single mutants. Controlling either ziaA or coaT with opposing regulators restored no resistance to metals sensed by the regulators, but coincident replacement of the deduced cytosolic amino-terminal domain CoaTN with ZiaAN (in ziaR-p¬†ziaA-ziaANcoaT) conferred Zn2+ resistance to ΔziaΔcoa, Zn2+ content was lowered and residual Co2+ resistance lost. Metal-dependent molar absorptivity under anaerobic conditions revealed that purified ZiaAN binds Co2+ in a pseudotetrahedral two-thiol site, and Co2+ was displaced by Zn2+. Thus, the amino-terminal domain of ZiaA inverts the metals exported by zinc-regulated CoaT from Co2+ to Zn2+, and this correlates simplistically with metal-binding preferences; KZiaAN Zn2+ tighter than Co2+. However, Zn2+ did not bleach Cu+-ZiaAN, and only Cu+ co-migrated with ZiaAN after competitive binding versus Zn2+. Bacterial two-hybrid assays that detected interaction between the Cu+-metallochaperone Atx1 and the amino-terminal domain of Cu+-transporter PacSN detected no interaction with the analogous, deduced, ferredoxin-fold subdomain of ZiaAN. Provided that there is no freely exchangeable cytosolic Cu+, restricted contact with the Cu+-metallochaperone can impose a barrier impairing the formation of otherwise favoured Cu+–ZiaAN complexes.
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Document Type: Research Article

Affiliations: Cell and Molecular Biosciences, Medical School, University of Newcastle, Newcastle upon Tyne NE2 4HH, UK.

Publication date: July 1, 2004

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