Borrelly Gilles P M, Rondet Sabine A M, Tottey Stephen, Robinson Nigel J
Cell and Molecular Biosciences, Medical School, University of Newcastle, Newcastle upon Tyne NE2 4HH, UK.
Mol Microbiol. 2004 Jul;53(1):217-27. doi: 10.1111/j.1365-2958.2004.04106.x.
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.
锌离子响应阻遏物ZiaR和钴离子响应激活物CoaR分别调节P(1)型锌离子（ZiaA）和钴离子（CoaT）ATP酶的产生。是什么决定了金属选择性？我们发现ΔziaΔcoa双突变体对锌离子的抗性与Δzia单突变体相似，对钴离子的抗性与Δcoa单突变体相似。用相反的调节因子控制ziaA或coaT都不能恢复对调节因子所感应金属的抗性，但将推导的胞质氨基末端结构域CoaT(N)与ZiaA(N)同时替换（在ziaR-(p) ziaA-ziaA(N)coaT中）赋予了ΔziaΔcoa对锌离子的抗性，降低了锌离子含量并丧失了残余的钴离子抗性。厌氧条件下的金属依赖性摩尔吸光率表明，纯化的ZiaA(N)在一个假四面体双硫醇位点结合钴离子，并且钴离子被锌离子取代。因此，ZiaA的氨基末端结构域将锌调节的CoaT输出的金属从钴离子转变为锌离子，这与金属结合偏好简单相关；K(ZiaAN)对锌离子的亲和力比对钴离子更强。然而，锌离子不会使铜离子-ZiaA(N)褪色，并且在与锌离子竞争结合后，只有铜离子与ZiaA(N)共同迁移。检测铜离子金属伴侣Atx1与铜离子转运蛋白PacS(N)的氨基末端结构域之间相互作用的细菌双杂交试验未检测到与ZiaA(N)类似的、推导的铁氧还蛋白折叠亚结构域的相互作用。假设不存在可自由交换的胞质铜离子，与铜离子金属伴侣的有限接触可能会形成一个障碍，损害原本有利的铜离子-ZiaA(N)复合物的形成。
