Stejskalova Camilla, Arrigoni Federica, Albanesi Riccardo, Bertini Luca, Mollica Luca, Coscia Francesca
Human Technopole, Structural Biology Centre, Milano, Italy.
Department of Biotechnology and Biosciences, University of Milano-Bicocca, Milano, Italy.
J Biol Chem. 2025 Jan;301(1):108026. doi: 10.1016/j.jbc.2024.108026. Epub 2024 Nov 26.
Thyroxine, the main hormone product of the thyroid, is produced at multiple sites within its protein precursor thyroglobulin. Each site consists of two tyrosine residues which undergo iodination and coupling, resulting in the synthesis of thyroxine at the acceptor tyrosine, where the hormone synthesis is later completed by proteolysis. Within the structurally resolved sites, the role of an essential conserved acidic residue preceding the acceptor remains elusive. To elucidate the mechanism of thyroxine synthesis we engineered a single-site minimal protein precursor. First, by its in vitro iodination and site-directed mutagenesis we show that the presence of the acidic residue, preferably glutamate, favors thyroxine synthesis. Secondly, within the designed precursor, we computationally modeled the reaction of iodination and iodotyrosine coupling giving rise to thyroxine. Our results reveal that hormone formation is triggered by iodotyrosine deprotonation, facilitated by proximity to a carboxylic group, closer in the case of glutamate, in line with our experimental findings and sequence conservation. Hereafter, we surmise that in the natural precursor thyroglobulin, two evolutionary late and slower hormonogenic sites coexist with an early evolutionary and faster one. Indeed, the latter is overlapping with a proteolytic site, thereby allowing prompt thyroxine release from thyroglobulin.
甲状腺素是甲状腺的主要激素产物，在其蛋白质前体甲状腺球蛋白的多个位点产生。每个位点由两个酪氨酸残基组成，它们会发生碘化和偶联反应，从而在受体酪氨酸处合成甲状腺素，随后通过蛋白水解完成激素合成。在结构解析的位点内，受体前一个必需的保守酸性残基的作用仍不清楚。为了阐明甲状腺素合成的机制，我们设计了一种单一位点的最小蛋白质前体。首先，通过体外碘化和定点诱变，我们发现酸性残基（最好是谷氨酸）的存在有利于甲状腺素的合成。其次，在设计的前体中，我们通过计算模拟了碘化和碘酪氨酸偶联生成甲状腺素的反应。我们的结果表明，激素的形成是由碘酪氨酸的去质子化引发的，靠近羧基会促进这一过程，谷氨酸的情况下距离更近，这与我们的实验结果和序列保守性一致。在此之后，我们推测在天然前体甲状腺球蛋白中，两个进化较晚且生成激素较慢的位点与一个进化较早且较快的位点共存。实际上，后者与一个蛋白水解位点重叠，从而使甲状腺素能够迅速从甲状腺球蛋白中释放出来。
