人類轉甲狀腺素蛋白A97S突變之結構與生物物理研究

Abstract

轉甲狀腺素蛋白 (Transthyretin or TTR) 是一種分子量大小為55 kDa的四聚體 (tetramer) 人類蛋白，其功能是在血液以及腦脊液中運輸甲狀腺素 (Thyroxine or T4)和全反式視網醇結合蛋白4 (holo-form retinol-binding protein 4)。轉甲狀腺素蛋白相關類澱粉沉積症 (ATTR) 是一群特徵為具有轉甲狀腺素變異體蛋白的澱粉樣纖維在細胞外累積所導致的疾病。在台灣，A97S是台灣的轉甲狀腺素蛋白相關類澱粉沉積症患者中鑑定出來的主要突變種。在本篇研究中，我們結合高解析度的A97S轉甲狀腺素蛋白的分子結構與生物化學和生物物理的數據，以證明97號位點的丙胺酸替換成絲胺酸會引起FG環 (FG-loop) 局部區域的波動，從而導致穩定態的四聚體不穩定化，形成單體種類的增加，進而增強纖維化的可能性。根據熱量計測的結果，A97S四聚體不穩定化在與天然穩定劑相互作用之下會發生顯著的變化，顯示出與野生型 (Wild-type或WT) 轉甲狀腺素相似結合能量模式。然而，儘管轉甲狀腺素變異體的分子內氫鍵網路 (intramolecular hydrogen-bonding interaction network) 部分重新排列，A97S轉甲狀腺素蛋白的四聚體中的FG環的靈活性 (flexibility) 仍相對較高。此外，A97S替換引起FG環的結構與動態變化，同時還輕微影響轉甲狀腺素蛋白與全反式視網醇結合蛋白4的複合體交界面，導致結合親和力減弱約五倍。此外引入的R104H變異體在熱力學上穩定了A97S/R104H雙變異體的四聚體結構，明顯抑制了澱粉樣生成級聯(cascade)。總而言之，我們的研究闡明了的澱粉樣A97S發生機制，證明了其對結構的不穩定作用，導致單體FG環的靈活性增加並調節澱粉樣纖維生成的動力學。

Transthyretin (TTR) is a 55 kDa tetrameric human protein and functions to transport thyroxine (T4) and holo-form retinol-binding protein 4 (holo-RBP4) in human plasma and cerebrospinal fluid. Transthyretin amyloidosis or ATTR is a group of diseases characterized by the extracellular accumulation of the amyloid fibrils containing TTR variants. In Taiwan, Ala97Ser (A97S) is the predominant mutation identified in Taiwanese ATTR patients. In this study, we combine atomic-resolution structural details of A97S-TTR with biophysical and biochemical data to demonstrate that the substitution of Ala-97 with serine induces fluctuations in the local region of the FG-loops, bringing about the destabilizing native-state tetramer, the increasing monomeric species, and thus the augmented fibrillization potential. Based on the calorimetric results, the destabilization of the A97S tetramer undergoes significant changes when interacting with native-state stabilizers, displaying binding energy patterns similar to that of wild-type (WT) TTR. However, despite the partial rearrangement of the intramolecular hydrogen-bonding networks in TTR variants, the flexibility of FG-loops in tetrameric A97S-TTR is still relatively higher. Furthermore, the A97S substitution induces structural and dynamic changes in the FG-loops, while also slightly affecting the complex interface of TTR and holo-form retinol-binding protein 4, resulting in an approximately five-fold decrease in the binding affinity. Besides, the introduced R104H variant thermodynamically stabilizes the quaternary structure of the A97S/R104H double mutant, significantly inhibiting the amyloidogenesis cascade. Collectively, our findings elucidate the molecular mechanism of amyloidogenic A97S, demonstrating its destabilizing effect on the TTR structure, leading to increased flexibility in the FG-loops of the monomer and modulation of the kinetics in amyloid fibrillization.