Groeneweg Stefan, Lima de Souza Elaine C, Meima Marcel E, Peeters Robin P, Visser W Edward, Visser Theo J
The Rotterdam Thyroid Center & Department of Internal Medicine, Erasmus Medical Center, 3015 CN, Rotterdam, The Netherlands.
Endocrinology. 2017 Oct 1;158(10):3292-3306. doi: 10.1210/en.2017-00082.
Monocarboxylate transporter 8 (MCT8) facilitates cellular uptake and efflux of thyroid hormone (TH). Mutations in MCT8 result in severe intellectual and motor disability known as the Allan-Herndon-Dudley syndrome (AHDS). Previous studies have provided valuable insights into the putative mechanism of substrate binding in the inward-open conformation, required for TH efflux. The current study aims to delineate the mechanism of substrate binding in the outward-open conformation, required for TH uptake. Extensive chemical modification and site-directed mutagenesis studies were used to guide protein homology modeling of MCT8 in the outward-open conformation. Arg271 and Arg445 were modified by phenylglyoxal, which was partially prevented in the presence of substrate. Substrate docking in our outward-open model suggested an important role for His192 and Arg445 in substrate binding. Interestingly, mutations affecting these residues have been identified in patients who have AHDS. In addition, our outward-open model predicted the location of Phe189, Met227, Phe279, Gly282, Phe287, and Phe501 at the substrate-binding center, and their Ala substitution differentially affected the apparent Vmax and Km of T3 transport, with F189A, F279A, and F287A showing the highest impact. Thus, here we present an MCT8 homology model in the outward-open conformation, which supports the important role of His192 and Arg445 in substrate docking and identifies critical residues at the putative substrate-binding center. Our findings provide insights into MCT8 structure and function, which add to our understanding of the pathogenic mechanism of mutations found in patients who have AHDS and can be used to screen for novel substrates and inhibitors.
单羧酸转运蛋白8（MCT8）促进甲状腺激素（TH）的细胞摄取和流出。MCT8突变会导致严重的智力和运动障碍，即艾伦 - 赫恩登 - 达德利综合征（AHDS）。先前的研究为TH流出所需的内向开放构象中底物结合的推定机制提供了有价值的见解。当前的研究旨在阐明TH摄取所需的外向开放构象中底物结合的机制。广泛的化学修饰和定点诱变研究被用于指导MCT8外向开放构象的蛋白质同源性建模。精氨酸271和精氨酸445被苯乙二醛修饰，在底物存在的情况下这种修饰被部分阻止。我们外向开放模型中的底物对接表明组氨酸192和精氨酸445在底物结合中起重要作用。有趣的是，在患有AHDS的患者中已鉴定出影响这些残基的突变。此外，我们的外向开放模型预测了苯丙氨酸189、甲硫氨酸227、苯丙氨酸279、甘氨酸282、苯丙氨酸287和苯丙氨酸501在底物结合中心的位置，它们的丙氨酸替代对T3转运的表观Vmax和Km有不同影响，其中F189A、F279A和F287A的影响最大。因此，在这里我们展示了一个MCT8外向开放构象的同源模型，该模型支持组氨酸192和精氨酸445在底物对接中的重要作用，并确定了推定底物结合中心的关键残基。我们的发现为MCT8的结构和功能提供了见解，这有助于我们理解在患有AHDS的患者中发现的突变的致病机制，并可用于筛选新型底物和抑制剂。
