Metallothioneins (MTs), small cysteine-rich proteins, present in four major isoforms, are key proteins involved in zinc and copper homeostasis in mammals. To date, only one X-ray crystal structure of a MT has been solved. It demonstrates seven bivalent metal ions bound in two structurally independent domains with M₄S₁₁ (α) and M₃S₉ (β) clusters. Recent discoveries indicate that Zn(II) ions are bound with MT2 with the range from nano- to picomolar affinity, which determines its cellular zinc buffering properties that are demonstrated by the presence of partially Zn(II)-depleted MT2 species. These forms serve as Zn(II) donors or acceptors and are formed under varying cellular free Zn(II) concentrations. Due to the lack of appropriate methods, knowledge regarding the structure of partially-depleted metallothionein is lacking. Here, we describe the Zn(II) binding mechanism in human MT2 with high resolution with respect to particular Zn(II) binding sites, and provide structural insights into Zn(II)-depleted MT species. The results were obtained by the labelling of metal-free cysteine residues with iodoacetamide and subsequent top-down electrospray ionization analysis, MALDI MS, bottom-up nanoLC-MALDI-MS/MS approaches and molecular dynamics (MD) simulations. The results show that the α-domain is formed sequentially in the first stages, followed by the formation of the β-domain, although both processes overlap, which is in contrast to the widely investigated cadmium MT. Independent ZnS₄ cores are characteristic for early stages of domain formation and are clustered in later stages. However, Zn–S network rearrangement in the β-domain upon applying the seventh Zn(II) ion explains its lower affinity. Detailed analysis showed that the weakest Zn(II) ion associates with the β-domain by coordination to Cys21, which was also found to dissociate first in the presence of the apo-form of sorbitol dehydrogenase. We found that Zn(II) binding to the isolated β-domain differs significantly from the whole protein, which explains its previously observed different Zn(II)-binding properties. MD results obtained for Zn(II) binding to the whole protein and isolated β-domain are highly convergent with mass spectrometry data. This study provides a comprehensive overview of the crosstalk of structural and zinc buffering related-to-thermodynamics properties of partially metal-saturated mammalian MT2 and sheds more light on other MT proteins and zinc homeostasis.

中文翻译：

---

哺乳动物金属硫蛋白-2 †的结构和锌缓冲特性的串扰

金属硫蛋白（MTs）是存在于四种主要同工型中的富含半胱氨酸的小蛋白，是哺乳动物中锌和铜稳态的关键蛋白。迄今为止，仅解决了MT的一种X射线晶体结构。它证明了在两个具有M ₄ S ₁₁（α）和M ₃ S ₉（β）簇的结构独立的域中结合的七个二价金属离子。最近的发现表明，Zn（II）离子与MT2结合的亲和力范围为纳摩尔至皮摩尔，这决定了其细胞锌缓冲特性，这通过部分Zn（II）耗尽的MT2物种的存在来证明。这些形式用作Zn（II）供体或受体，并在不同的细胞游离Zn（II）浓度下形成。由于缺乏适当的方法，因此缺乏有关部分耗尽的金属硫蛋白结构的知识。在这里，我们描述了锌（II）在高分辨率的人类MT2绑定机制相对于特定的Zn（II）的结合位点，并提供结构见解锌（II）耗尽的MT物种。通过使用碘乙酰胺标记无金属的半胱氨酸残基，然后进行自上而下的电喷雾电离分析，MALDI MS，自下而上的nanoLC-MALDI-MS / MS方法和分子动力学（MD）模拟，可获得结果。结果表明，尽管两个过程重叠，但在第一阶段中依次形成了α-畴，然后形成了β-畴，这与广泛研究的镉MT相反。独立的ZnS ₄核是畴形成早期的特征，并在后期聚集。但是，在施加第七个Zn（II）离子后，Zn-S网络在β区的重排说明了其较低的亲和力。详细分析表明，最弱的Zn（II）离子通过与Cys21配位而与β结构域缔合，而Cys21在山梨糖醇脱氢酶脱辅基形式的存在下也首先解离。我们发现，Zn（II）与分离的β结构域的结合与整个蛋白质有显着差异，这说明了其先前观察到的Zn（II）结合特性不同。Zn（II）与整个蛋白质和分离的β结构域结合获得的MD结果与质谱数据高度一致。这项研究提供了对部分金属饱和的哺乳动物MT2的结构和锌缓冲与热力学性质的串扰的全面概述，并为其他MT蛋白和锌稳态提供了更多的信息。