Sutherland Duncan E K, Stillman Martin J
Department of Chemistry, The University of Western Ontario, London, ON, Canada.
Metallomics. 2014 Apr;6(4):702-28. doi: 10.1039/c3mt00216k.
Metallothioneins (MT) are a family of small cysteine rich proteins that have been implicated in a range of roles including toxic metal detoxification, protection against oxidative stress, and as metallochaperones are undoubtedly involved in the homeostasis of both essential zinc and copper. While complete details of all possible cellular functions are still unknown, it is clear that they must be directly related to both the accessibility and the metal-binding properties of the many cysteine residues in the protein. The most well studied MTs are of mammalian origin and consist of two domains: a β-domain with 9 cysteine residues that sequesters 3 Cd(2+), 3 Zn(2+) or 6 Cu(+) ions, and an α-domain with 11 cysteine residues that sequesters 4 Cd(2+), 4 Zn(2+) or 6 Cu(+) ions. The key to understanding the cellular importance of MT in these different roles is in a precise description of the metallation status before and during reactions. An assessment of all possible and all biologically accessible metallation states is necessary before the functional mechanistic details can be fully determined. Conventionally, it has been considered that metal ions bind in a domain-specific and, therefore, cooperative manner, where the apparently isolated domains fill with their full complement of metal ions immediately with no discernible or measurable intermediates. A number of detailed mechanistic studies of the metallation reactions of mammalian MTs have provided significant insight into the metallation reactions. Recent results from electrospray ionization mass spectrometric studies of the stepwise metallation of the two fragments and the whole protein with Zn(2+), Cd(2+), As(3+) and Bi(3+) indicate a noncooperative mechanism of a declining series of K(F)'s. Of particular note are new details about the early stages of the stepwise metallation reactions, specifically the stability of partially metallated species for As(3+), Cd(2+), and Zn(2+) that do not correspond to the two-domain model. In addition, at the other end of the coordination spectrum are the supermetallated species of MT, where supermetallation defines metallation in excess of traditional levels. It has been reported that with metal ion excess the formation of a single 'super domain' is possible and again this deviates from the two-domain model of MT. In both cases, these results suggest that the structural view of mammalian MT that is of two essentially isolated domains may be the exceptional case and that under the normal conditions of cellular metal-ion concentrations the two domain structure might coexist in equilibrium with various single domain, multi-metal site structures. This review specifically focuses on providing context for these recent studies and the new ideas concerning metallation prior to the establishment of domain-based clusters that these studies suggest.
金属硫蛋白（MT）是一类富含半胱氨酸的小分子蛋白质家族，其作用广泛，包括对有毒金属的解毒、抵御氧化应激，并且作为金属伴侣无疑参与了必需锌和铜的体内稳态。虽然所有可能的细胞功能的完整细节仍不明确，但很明显它们必定与蛋白质中许多半胱氨酸残基的可及性和金属结合特性直接相关。研究最为深入的MT来源于哺乳动物，由两个结构域组成：一个β结构域有9个半胱氨酸残基，可螯合3个Cd(2+)、3个Zn(2+)或6个Cu(+)离子；一个α结构域有11个半胱氨酸残基，可螯合4个Cd(2+)、4个Zn(2+)或6个Cu(+)离子。理解MT在这些不同作用中细胞重要性的关键在于精确描述反应前后的金属化状态。在能够完全确定功能机制细节之前，评估所有可能的和所有生物学上可及的金属化状态是必要的。传统上，人们认为金属离子以结构域特异性且因此是协同的方式结合，即明显孤立的结构域会立即充满其全部金属离子，没有可辨别的或可测量的中间体。对哺乳动物MT金属化反应的一些详细机制研究为金属化反应提供了重要见解。最近通过电喷雾电离质谱对两个片段和整个蛋白质与Zn(2+)、Cd(2+)、As(3+)和Bi(3+)的逐步金属化研究结果表明，其机制是非协同的，K(F)呈递减序列。特别值得注意的是关于逐步金属化反应早期阶段的新细节，具体而言是As(3+)、Cd(2+)和Zn(2+)部分金属化物种的稳定性，这与双结构域模型不符。此外，在配位谱的另一端是MT的超金属化物种，其中超金属化定义为超过传统水平的金属化。据报道，在金属离子过量时，可能形成单个“超结构域”，这同样偏离了MT的双结构域模型。在这两种情况下，这些结果表明哺乳动物MT的两个基本孤立结构域的结构观点可能是例外情况，并且在细胞金属离子浓度的正常条件下，双结构域结构可能与各种单结构域、多金属位点结构处于平衡共存状态。本综述特别着重于为这些近期研究以及这些研究提出的在基于结构域的簇形成之前有关金属化的新观点提供背景信息。
