Structure and metal loading of a soluble periplasm cuproprotein.
Structure and metal loading of a soluble periplasm cuproprotein.
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可溶性周质铜蛋白的结构和金属负载。
DOI：
10.1074/jbc.m110.153080
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发表时间：
2010
期刊：
影响因子：
--
通讯作者：
Waldron KJ
中科院分区：
文献类型：
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作者：
Waldron KJ
研究发现，即使周质中存在丰富的竞争性 Zn2+，铜运输途径也能使 Cu2+ 占据可溶性周质蛋白 CucA。在这里，我们解析了 CucA（一种新的 cupin）的结构，发现 Cu2+（而不是 Zn2+）的结合会猝灭与金属位点相邻的 Trp165 的荧光。使用这种荧光探针，我们确定在暴露于等摩尔的 Zn2+ 和 Cu2+ 后，CucA 部分被 Zn2+ 占据。 Cu2+-CucA 比 Zn2+-CucA 更热力学稳定，但 (Zn→Cu) 交换很慢，这引发了关于周质如何仅包含 Cu2+ 形式的问题。我们发现涉及两个铜转运蛋白（CtaA 和 PacS）和金属伴侣（Atx1）的铜运输途径对于 Cu2+-CucA 在周质中积累是必需的。 ΔctaA细胞周质中的CucA蛋白可以忽略不计，但cucA转录本的丰度没有改变。至关重要的是，ΔctaA细胞在周质中过度积累低Mrcopper复合物，纯化的apoCucA可以很容易地从ΔctaAperiplasm提取物中获取Cu2+，但在体内apoCucA无法与这些周质铜池接触。相反，铜通过细胞质途径运输，该途径与 CucA 易位至周质相结合。
A copper-trafficking pathway was found to enable Cu2+occupancy of a soluble periplasm protein, CucA, even when competing Zn2+is abundant in the periplasm. Here, we solved the structure of CucA (a new cupin) and found that binding of Cu2+, but not Zn2+, quenches the fluorescence of Trp165, which is adjacent to the metal site. Using this fluorescence probe, we established that CucA becomes partly occupied by Zn2+following exposure to equimolar Zn2+and Cu2+. Cu2+-CucA is more thermodynamically stable than Zn2+-CucA butk(Zn→Cu)exchangeis slow, raising questions about how the periplasm contains solely the Cu2+form. We discovered that a copper-trafficking pathway involving two copper transporters (CtaA and PacS) and a metallochaperone (Atx1) is obligatory for Cu2+-CucA to accumulate in the periplasm. There was negligible CucA protein in the periplasm of ΔctaAcells, but the abundance ofcucAtranscripts was unaltered. Crucially, ΔctaAcells overaccumulate lowMrcopper complexes in the periplasm, and purified apoCucA can readily acquire Cu2+from ΔctaAperiplasm extracts, butin vivoapoCucA fails to come into contact with these periplasmic copper pools. Instead, copper traffics via a cytoplasmic pathway that is coupled to CucA translocation to the periplasm.