Departments of Biochemistry and Nutritional Sciences, School of Life Course Sciences, Faculty of Life Sciences and Medicine, King's College London, Franklin-Wilkins Bldg, 150 Stamford St., London SE1 9NH, UK.
Department of Analytical Chemistry, Faculty of Pharmacy in Hradec Králové, Charles University, Heyrovského 1203, 500 05 Hradec Králové, Czech Republic.
Int J Mol Sci. 2021 Mar 14;22(6):2940. doi: 10.3390/ijms22062940.
The human zinc transporter ZnT8 provides the granules of pancreatic β-cells with zinc (II) ions for assembly of insulin hexamers for storage. Until recently, the structure and function of human ZnTs have been modelled on the basis of the 3D structures of bacterial zinc exporters, which form homodimers with each monomer having six transmembrane α-helices harbouring the zinc transport site and a cytosolic domain with an α,β structure and additional zinc-binding sites. However, there are important differences in function as the bacterial proteins export an excess of zinc ions from the bacterial cytoplasm, whereas ZnT8 exports zinc ions into subcellular vesicles when there is no apparent excess of cytosolic zinc ions. Indeed, recent structural investigations of human ZnT8 show differences in metal binding in the cytosolic domain when compared to the bacterial proteins. Two common variants, one with tryptophan (W) and the other with arginine (R) at position 325, have generated considerable interest as the R-variant is associated with a higher risk of developing type 2 diabetes. Since the mutation is at the apex of the cytosolic domain facing towards the cytosol, it is not clear how it can affect zinc transport through the transmembrane domain. We expressed the cytosolic domain of both variants of human ZnT8 and have begun structural and functional studies. We found that (i) the metal binding of the human protein is different from that of the bacterial proteins, (ii) the human protein has a C-terminal extension with three cysteine residues that bind a zinc(II) ion, and (iii) there are small differences in stability between the two variants. In this investigation, we employed nickel(II) ions as a probe for the spectroscopically silent Zn(II) ions and utilised colorimetric and fluorimetric indicators for Ni(II) ions to investigate metal binding. We established Ni(II) coordination to the C-terminal cysteines and found differences in metal affinity and coordination in the two ZnT8 variants. These structural differences are thought to be critical for the functional differences regarding the diabetes risk. Further insight into the assembly of the metal centres in the cytosolic domain was gained from potentiometric investigations of zinc binding to synthetic peptides corresponding to N-terminal and C-terminal sequences of ZnT8 bearing the metal-coordinating ligands. Our work suggests the involvement of the C-terminal cysteines, which are part of the cytosolic domain, in a metal chelation and/or acquisition mechanism and, as now supported by the high-resolution structural work, provides the first example of metal-thiolate coordination chemistry in zinc transporters.
人类锌转运蛋白 ZnT8 为胰腺β细胞的颗粒提供锌 (II) 离子，用于胰岛素六聚体的组装和储存。直到最近，人们一直基于细菌锌外排蛋白的 3D 结构来模拟人类 ZnTs 的结构和功能，这些蛋白形成同源二聚体，每个单体具有六个跨膜α-螺旋，包含锌转运位点和一个具有α、β结构和额外锌结合位点的胞质域。然而，在功能上存在重要差异，因为细菌蛋白从细菌细胞质中排出过量的锌离子，而 ZnT8 在细胞质中没有明显过量的锌离子时，将锌离子输出到亚细胞囊泡中。事实上，最近对人类 ZnT8 的结构研究表明，与细菌蛋白相比，在胞质域中金属结合存在差异。两个常见的变体，一个在 325 位有色氨酸 (W)，另一个有精氨酸 (R)，引起了相当大的兴趣，因为 R 变体与患 2 型糖尿病的风险增加有关。由于突变位于面向细胞质的胞质域的顶点，因此不清楚它如何影响跨膜域的锌转运。我们表达了人类 ZnT8 的两种变体的胞质域，并开始进行结构和功能研究。我们发现：(i) 人类蛋白的金属结合与细菌蛋白不同；(ii) 人类蛋白有一个 C 末端延伸，带有三个半胱氨酸残基，可结合一个锌 (II) 离子；(iii) 两种变体之间存在稳定性的微小差异。在这项研究中，我们使用镍 (II) 离子作为光谱上无声的 Zn(II) 离子的探针，并利用比色和荧光指示剂来研究 Ni(II) 离子的结合。我们确定了 Ni(II) 与 C 末端半胱氨酸的配位，并发现了两种 ZnT8 变体在金属亲和力和配位方面的差异。这些结构差异被认为是与糖尿病风险相关的功能差异的关键。通过对合成肽进行电位测定，研究了锌与 ZnT8 的 N 末端和 C 末端序列对应物的结合，进一步深入了解了胞质域中金属中心的组装情况，这些肽带有金属配位配体。我们的工作表明，C 末端半胱氨酸参与了金属螯合和/或获取机制，并且现在得到高分辨率结构工作的支持，提供了锌转运蛋白中金属-硫醇配位化学的第一个例子。
