title: Chimeras of P-type ATPases and their transcriptional regulators: contributions of a cytosolic amino-terminal domain to metal specificity.
abstract: Zn(2+)-responsive repressor ZiaR and Co(2+)-responsive activator CoaR modulate production of P(1)-type Zn(2+)- (ZiaA) and Co(2+)- (CoaT) ATPases respectively. What dictates metal selectivity? We show that Delta ziaDeltacoa double mutants had similar Zn(2+) resistance to Deltazia single mutants and similar Co(2+) 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 Zn(2+) resistance to DeltaziaDeltacoa, Zn(2+) content was lowered and residual Co(2+) resistance lost. Metal-dependent molar absorptivity under anaerobic conditions revealed that purified ZiaA(N) binds Co(2+) in a pseudotetrahedral two-thiol site, and Co(2+) was displaced by Zn(2+). Thus, the amino-terminal domain of ZiaA inverts the metals exported by zinc-regulated CoaT from Co(2+) to Zn(2+), and this correlates simplistically with metal-binding preferences; K(ZiaAN) Zn(2+) tighter than Co(2+). However, Zn(2+) did not bleach Cu(+)-ZiaA(N), and only Cu(+) co-migrated with ZiaA(N) after competitive binding versus Zn(2+). 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.
authors.affiliation: Cell and Molecular Biosciences, Medical School, University of Newcastle, Newcastle upon Tyne NE2 4HH, UK.
authors.forename: Gilles P M
authors.initials: GP
authors.lastname: Borrelly
authors.forename: Sabine A M
authors.initials: SA
authors.lastname: Rondet
authors.forename: Stephen
authors.initials: S
authors.lastname: Tottey
authors.forename: Nigel J
authors.initials: NJ
authors.lastname: Robinson
mesh_terms: D000251:Adenosine Triphosphatases / Q000235:genetics / Q000378:metabolism*; D000458:Cyanobacteria / Q000201:enzymology / Q000235:genetics*; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D014158:Transcription, Genetic; D015032:Zinc / Q000378:metabolism
chemical_list: D011993:Recombinant Fusion Proteins; D000251:Adenosine Triphosphatases; C109898:Zn(II)-translocating P-type ATPase; D015032:Zinc