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A transthyretin monomer intermediate undergoes local unfolding and transient interaction with oligomers in a kinetically concerted aggregation pathway
Journal of Biological Chemistry ( IF 5.5 ) Pub Date : 2022-06-18 , DOI: 10.1016/j.jbc.2022.102162
Xun Sun 1 , James A Ferguson 1 , H Jane Dyson 1 , Peter E Wright 1
Affiliation  

Transthyretin (TTR) amyloidosis is associated with tissue deposition of TTR aggregates. TTR aggregation is initiated by dissociation of the native tetramer to form a monomeric intermediate, which locally unfolds and assembles into soluble oligomers and higher-order aggregates. However, a detailed mechanistic understanding requires kinetic and structural characterization of the low population intermediates formed. Here, we show that the monomeric intermediate exchanges with an ensemble of oligomers on the millisecond timescale. This transient and reversible exchange causes broadening of the 19F resonance of a trifluoromethyl probe coupled to the monomeric intermediate at S85C. We show the 19F linewidth and R2 relaxation rate increase with increasing concentration of the oligomer. Furthermore, introduction of 19F probes at additional TTR sites yielded distinct 19F chemical shifts for the TTR tetramer and monomer when the trifluoromethyl probe was attached at S100C, located near the same subunit interface as S85C, but not with probes attached at S46C or E63C, which are distant from any interfaces. The 19F probe at E63C shows that part of the DE loop, which is solvent accessible in the tetramer, becomes more buried in the NMR-visible oligomers. Finally, using backbone amides as probes, we show that parts of the EF helix and H-strand become highly flexible in the otherwise structured monomeric intermediate at acidic pH. We further find that TTR aggregation can be reversed by increasing pH. Taken together, this work provides insights into location-dependent conformational changes in the reversible early steps of a kinetically concerted TTR aggregation pathway.



中文翻译:

转甲状腺素蛋白单体中间体在动力学协同聚集途径中与低聚物发生局部展开和瞬时相互作用

转甲状腺素蛋白 (TTR) 淀粉样变性与 TTR 聚集体的组织沉积有关。TTR 聚集是由天然四聚体解离形成单体中间体开始的,该中间体在局部展开并组装成可溶性低聚物和高阶聚集体。然而,详细的机理理解需要对形成的低种群中间体进行动力学和结构表征。在这里,我们展示了单体中间体在毫秒时间尺度上与一组低聚物交换。这种瞬时和可逆的交换导致在 S85C 与单体中间体偶联的三氟甲基探针的19 F 共振变宽。我们展示了19 F 线宽和R 2弛豫率随着低聚物浓度的增加而增加。此外,当三氟甲基探针附着在S100C (与S85C位于相同的亚基界面附近,但没有探针附着在 S46C 或E63C,它们远离任何接口。19 _E63C 处的 F 探针显示,在四聚体中溶剂可接近的部分 DE 环变得更加埋藏在 NMR 可见的低聚物中。最后,使用主链酰胺作为探针,我们发现部分 EF 螺旋和 H 链在酸性 pH 条件下在其他结构化的单体中间体中变得高度灵活。我们进一步发现 TTR 聚集可以通过增加 pH 值来逆转。总之,这项工作提供了对动力学协同 TTR 聚集途径的可逆早期步骤中位置依赖性构象变化的见解。

更新日期:2022-06-18
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