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

Zn2+-responsive repressor ZiaR and Co2+-responsive activator CoaR modulate production of P-1-type Zn2+- (ZiaA) and Co2+- (CoaT) ATPases respectively. What dictates metal selectivity? We show that Delta ziaDeltacoa double mutants had similar Zn2+ resistance to Deltazia single mutants and similar Co2+ resistance to Deltacoa 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 CoaT(N) with ZiaA(N) (in ziaR-(p) ziaA-ziaA(N)coaT) conferred Zn2+ resistance to DeltaziaDeltacoa, Zn2+ content was lowered and residual Co2+ resistance lost. Metal-dependent molar absorptivity under anaerobic conditions revealed that purified ZiaA(N) 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; K-ZiaAN Zn2+ tighter than Co2+. However, Zn2+ did not bleach Cu+-ZiaA(N), and only Cu+ co-migrated with ZiaA(N) 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 PacS(N) detected no interaction with the analogous, deduced, ferredoxin-fold subdomain of ZiaA(N). 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+-ZiaA(N) complexes.