Institute of Crystallography, National Council of Research, CNR, S.S. Catania, Via P. Gaifami 18, 95126 Catania, Italy.
Consorzio Interuniversitario per la Ricerca dei Metalli nei Sistemi Biologici, Via Ulpiani 27, 70126 Bari, Italy.
Int J Mol Sci. 2022 Mar 8;23(6):2929. doi: 10.3390/ijms23062929.
Ctr1 regulates copper uptake and its intracellular distribution. The first 14 amino acid sequence of the Ctr1 ectodomain Ctr1 encompasses the characteristic Amino Terminal Cu and Ni binding motif (ATCUN) as well as the bis-His binding motif (His5 and His6). We report a combined thermodynamic and spectroscopic (UV-vis, CD, EPR) study dealing with the formation of Cu homobinuclear complexes with Ctr1, the percentage of which is not negligible even in the presence of a small Cu excess and clearly prevails at a M/L ratio of 1.9. Ascorbate fails to reduce Cu when bound to the ATCUN motif, while it reduces Cu when bound to the His5-His6 motif involved in the formation of binuclear species. The histidine diade characterizes the second binding site and is thought to be responsible for ascorbate oxidation. Binding constants and speciation of Ag complexes with Ctr1, which are assumed to mimic Cu interaction with N-terminus of Ctr1, were also determined. A preliminary immunoblot assay evidences that the anti-Ctr1 extracellular antibody recognizes Ctr1 in a different way from the longer Ctr1 that encompasses a second His and Met rich domain.
Ctr1 调节铜的摄取及其细胞内分布。Ctr1 外域的前 14 个氨基酸序列包含特征性的氨基末端 Cu 和 Ni 结合基序（ATCUN）以及双组氨酸结合基序（His5 和 His6）。我们报告了一项联合热力学和光谱学（UV-vis、CD、EPR）研究，涉及 Ctr1 形成 Cu 同核双核配合物的情况，即使在存在少量 Cu 过剩的情况下，其形成的百分比也不容忽视，并且在 M/L 比为 1.9 时明显占优势。当与 ATCUN 基序结合时，抗坏血酸不能还原 Cu，但当与参与双核物种形成的 His5-His6 基序结合时，抗坏血酸可以还原 Cu。组氨酸二联体特征是第二个结合位点，被认为负责抗坏血酸的氧化。还确定了 Ctr1 与 Ag 配合物的结合常数和形态，假设它们模拟 Cu 与 Ctr1 N 末端的相互作用。初步的免疫印迹分析表明，抗 Ctr1 细胞外抗体与包含第二个 His 和 Met 丰富结构域的更长的 Ctr1 以不同的方式识别 Ctr1。
