Transthyretin (TTR) is an homotetrameric protein involved in the transport of thyroxine. More than 150 different mutations have been described in the TTR gene, several of them associated with familial amyloid cardiomyopathy. Recently, our group described a new variant of TTR in Brazil, namely A39D-TTR, which causes a severe cardiac condition. Position 39 is in the AB loop, a region of the protein that is located within the thyroxine-binding channels and is involved in tetramer formation. In the present study, we solved the structure and characterize the thermodynamic stability of this new variant of TTR using urea and high hydrostatic pressure. Interestingly, during the process of purification, A39D-TTR turned out to be a dimer and not a tetramer, a variation that might be explained by the close contact of the four aspartic acids at position 39, where they face each other inside the thyroxine channel. In the presence of subdenaturing concentrations of urea, bis-ANS binding and dynamic light scattering revealed A39D-TTR in the form of a molten-globule dimer. Co-expression of A39D and WT isoforms in the same bacterial cell did not produce heterodimers or heterotetramers, suggesting that somehow a negative charge at the AB loop precludes tetramer formation. A39D-TTR proved to be highly amyloidogenic, even at mildly acidic pH values where WT-TTR does not aggregate. Interestingly, despite being a dimer, aggregation of A39D-TTR was inhibited by diclofenac, which binds to the thyroxine channel in the tetramer, suggesting the existence of other pockets in A39D-TTR able to accommodate this molecule.

中文翻译：

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参与严重心肌病的转甲状腺素蛋白高度淀粉样蛋白生成二聚体的结构和热力学特征


转甲状腺素运载蛋白 （TTR） 是一种参与甲状腺素转运的同源四聚体蛋白。TTR 基因中已描述 150 多种不同的突变，其中几种与家族性淀粉样变性心肌病有关。最近，我们小组在巴西描述了一种新的 TTR 变体，即 A39D-TTR，它会导致严重的心脏病。位置 39 位于 AB 环中，这是蛋白质的一个区域，位于甲状腺素结合通道内，参与四聚体形成。在本研究中，我们使用尿素和高静水压力解决了 TTR 的这种新变体的结构并表征了热力学稳定性。有趣的是，在纯化过程中，A39D-TTR 被证明是二聚体而不是四聚体，这种变化可能是由于四种天冬氨酸在第 39 位的紧密接触，它们在甲状腺素通道内彼此相对。在亚变性浓度尿素存在下，双 ANS 结合和动态光散射显示 A39D-TTR 以熔融小球二聚体的形式存在。A39D 和 WT 亚型在同一细菌细胞中的共表达不会产生异二聚体或异四聚体，这表明 AB 环上的负电荷以某种方式排除了四聚体的形成。A39D-TTR 被证明具有高度淀粉样蛋白生成性，即使在 WT-TTR 不聚集的弱酸性 pH 值下也是如此。有趣的是，尽管 A39D-TTR 是二聚体，但 A39D-TTR 的聚集被双氯芬酸抑制，双氯芬酸与四聚体中的甲状腺素通道结合，表明 A39D-TTR 中存在能够容纳该分子的其他口袋。