Food Chemistry and Toxicology, Institute for Applied Biosciences, Karlsruhe Institute of Technology (KIT), Kaiserstrasse 12, 76131 Karlsruhe, Germany.
Metallomics. 2019 May 22;11(5):968-981. doi: 10.1039/c8mt00308d.
Human serum transferrin (hTF) is an iron binding protein with the primary task of ensuring well-controlled transport of Fe3+-ions in the bloodstream. Furthermore, hTF has been identified as a key component in the trafficking of Al3+-ions from the serum to cells. It is clear that binding alone does not guarantee cellular uptake via the transferrin receptor, since this is determined by the structural properties of the metal-protein complex. The conformation of the metallated hTF is critically important for delivery of Fe3+ or any other metal into the cell. The combination of ultra-high resolution ESI mass spectrometry and CD spectroscopy together provide accurate species distribution of the Fe3+ during stepwise addition to apo-hTF and an indirect indication of the tertiary structure of the metallated protein. These two methods together are extremely fine probes of structural changes as a function of precise metal binding status at micromolar concentrations. Simulation of the precise domain distribution could be determined during the stepwise metallation from 0 to 2 Fe3+ added. Analysis of the ESI-MS data for the stepwise metallation of apo-hTF and Al1 or 2-hTF with Fe3+ was carried out and used to simulate the experimental speciation based on the reported KF values. There are six main conclusions: (1) Fe3+ binds predominantly, initially to the C-lobe. (2) The CD spectral properties indicate that the C-lobe metallation dominates the structural properties of both binding sites; N-lobe metallation modifies the C-lobe structure. (3) Fe3+ metallation of the mixed Al1-2-hTF results in the dominant form of Fe1Al1-hTF. (4) The first Fe3+ bound to Al1-hTF binds predominantly in the C-lobe domain. (5) The CD spectral properties when Fe3+ binds to Al1-2-hTF indicates that Al-N-lobe occupation mirrors the structural effects of N-lobe occupation by Fe3+. (6) With respect to how Al3+ might enter the cell, the formation of a hybrid form Al1Fe1-hTF might enable the Al3+ to enter the cell via receptor-mediated endocytosis due to the binding of Fe3+ in the C-lobe of the protein which is primarily responsible for the structure of the metal-protein complex.
人血清转铁蛋白（hTF）是一种铁结合蛋白，主要任务是确保血液中 Fe3+离子的良好控制运输。此外，hTF 已被确定为将 Al3+离子从血清转运到细胞的关键组成部分。显然，仅结合本身并不能保证通过转铁蛋白受体进行细胞摄取，因为这取决于金属-蛋白复合物的结构特性。金属化 hTF 的构象对于将 Fe3+或任何其他金属递送到细胞中至关重要。超高分辨率 ESI 质谱和 CD 光谱的组合一起提供了逐步添加到 apo-hTF 时 Fe3+的准确物种分布，并间接表明了金属化蛋白的三级结构。这两种方法一起是研究结构变化的极好探针，因为它们可以在微摩尔浓度下精确地反映金属结合状态。可以在逐步金属化过程中从 0 到 2 个 Fe3+添加来确定精确的结构域分布的模拟。对 apo-hTF 和 Al1 或 2-hTF 与 Fe3+的逐步金属化的 ESI-MS 数据进行了分析，并根据报道的 KF 值用于模拟实验物种形成。有六个主要结论：（1）Fe3+主要最初结合到 C lobe。（2）CD 光谱特性表明，C lobe 金属化主导两个结合位点的结构特性；N lobe 金属化修饰 C lobe 结构。（3）混合 Al1-2-hTF 的 Fe3+金属化导致 Fe1Al1-hTF 的主要形式。（4）第一个结合到 Al1-hTF 的 Fe3+主要结合到 C lobe 结构域。（5）当 Fe3+结合到 Al1-2-hTF 时，CD 光谱特性表明，Al-N lobe 占据反映了 N lobe 占据对 Fe3+的结构影响。（6）就 Al3+如何进入细胞而言，由于蛋白 C lobe 中 Fe3+的结合，形成混合形式的 Al1Fe1-hTF 可能使 Al3+通过受体介导的内吞作用进入细胞，因为蛋白 C lobe 主要负责金属-蛋白复合物的结构。
