Department of Chemistry, University of Toronto, Toronto, Ontario, Canada M5S 3H6.
J Am Chem Soc. 2009 Dec 30;131(51):18489-500. doi: 10.1021/ja9081765.
Metallochaperones are essential for the safe and targeted delivery of necessary yet toxic metal cofactors to their respective protein partners. In this study we examine the nickel-binding properties of the Escherichia coli protein SlyD, a factor that contributes to optimal nickel accumulation in this organism. This protein is also required for E. coli energy metabolism because it participates in the nickel insertion step during [Ni-Fe]-hydrogenase metallocenter assembly. Our study demonstrates that SlyD is a multiple nickel ion binding protein. The analysis of noncovalent metal-protein complexes via electrospray ionization mass spectrometry revealed that SlyD binds up to seven nickel ions in a noncooperative manner with submicromolar affinity (<2 microM, upper limit) and that the protein exists in a dynamic mixture of metalloforms that is dependent on the availability of nickel ions in solution. Structural analysis indicates that this metallochaperone undergoes small but distinct changes in the structure upon metal binding and that the nickel-binding sites are assembled through beta-turn formation. Although the C-terminal metal-binding domain is primarily responsible for metal chelation, we find that metal binding also perturbs the structure of the N-terminal domains. An investigation of the nickel sites by using X-ray absorption spectroscopy shows that SlyD binds nickel ions by adapting several different geometries and coordination numbers. Finally, the characterization of SlyD mutants demonstrates that the cysteine residues in the C-terminal domain confer tighter affinity as well as increased binding capacity to SlyD. On the basis of the presented data a model for nickel binding to SlyD as well as its role in nickel homeostasis is discussed.
金属伴侣蛋白对于将必需但有毒的金属辅因子安全且靶向递送到其各自的蛋白质伴侣中至关重要。在这项研究中，我们研究了大肠杆菌蛋白 SlyD 的镍结合特性，该蛋白有助于该生物体中最佳的镍积累。该蛋白还参与大肠杆菌的能量代谢，因为它参与 [Ni-Fe]-氢化酶金属中心组装过程中的镍插入步骤。我们的研究表明 SlyD 是一种多镍离子结合蛋白。通过电喷雾电离质谱分析非共价金属-蛋白质复合物表明，SlyD 以非协同方式结合多达七个镍离子，亲和力为亚微摩尔级（<2 microM，上限），并且该蛋白存在于依赖于溶液中镍离子可用性的动态金属形式混合物中。结构分析表明，这种金属伴侣蛋白在金属结合时会发生微小但明显的结构变化，并且镍结合位点通过β-转角形成组装。尽管 C 末端金属结合结构域主要负责金属螯合，但我们发现金属结合也会干扰 N 末端结构域的结构。通过使用 X 射线吸收光谱对镍结合位点的研究表明，SlyD 通过适应几种不同的几何形状和配位数来结合镍离子。最后，对 SlyD 突变体的表征表明，C 末端结构域中的半胱氨酸残基赋予 SlyD 更紧密的亲和力和更高的结合容量。根据所提供的数据，讨论了 SlyD 与镍结合及其在镍稳态中的作用的模型。
