Department of Experimental and Clinical Biomedical Sciences "Mario Serio", University of Florence, 50134 Florence, Italy.
Department of Medical Area, University of Udine, 33100 Udine, Italy.
Molecules. 2022 Jul 5;27(13):4309. doi: 10.3390/molecules27134309.
TAR DNA-binding protein 43 (TDP-43) forms intraneuronal cytoplasmic inclusions associated with amyotrophic lateral sclerosis and ubiquitin-positive frontotemporal lobar degeneration. Its N-terminal domain (NTD) can dimerise/oligomerise with the head-to-tail arrangement, which is essential for function but also favours liquid-liquid phase separation and inclusion formation of full-length TDP-43. Using various biophysical approaches, we identified an alternative conformational state of NTD in the presence of Sulfobetaine 3-10 (SB3-10), with higher content of α-helical structure and tryptophan solvent exposure. NMR shows a highly mobile structure, with partially folded regions and β-sheet content decrease, with a concomitant increase of α-helical structure. It is monomeric and reverts to native oligomeric NTD upon SB3-10 dilution. The equilibrium GdnHCl-induced denaturation shows a cooperative folding and a somewhat lower conformational stability. When the aggregation processes were compared with and without pre-incubation with SB3-10, but at the identical final SB3-10 concentration, a slower aggregation was found in the former case, despite the reversible attainment of the native conformation in both cases. This was attributed to protein monomerization and oligomeric seeds disruption by the conditions promoting the alternative conformation. Overall, the results show a high plasticity of TDP-43 NTD and identify strategies to monomerise TDP-43 NTD for methodological and biomedical applications.
TAR DNA 结合蛋白 43（TDP-43）形成与肌萎缩性侧索硬化症和泛素阳性额颞叶变性相关的神经元内细胞质包含物。其 N 端结构域（NTD）可以通过头对头排列形成二聚体/寡聚体，这对于功能至关重要，但也有利于全长 TDP-43 的液-液相分离和包含物形成。使用各种生物物理方法，我们在存在磺基甜菜碱 3-10（SB3-10）的情况下鉴定了 NTD 的替代构象状态，其具有更高含量的α-螺旋结构和色氨酸溶剂暴露。NMR 显示出高度移动的结构，部分折叠区域和β-折叠含量减少，同时α-螺旋结构增加。它是单体的，并在 SB3-10 稀释后恢复为天然寡聚 NTD。平衡 GdnHCl 诱导的变性显示出协同折叠和稍低的构象稳定性。当比较有和没有与 SB3-10 预孵育的聚合过程，但在相同的最终 SB3-10 浓度下，在前者中发现聚合速度较慢，尽管在两种情况下都可以可逆地达到天然构象。这归因于通过促进替代构象的条件使蛋白质单体化和寡聚体种子破坏。总体而言，结果表明 TDP-43 NTD 具有高度的可塑性，并确定了用于方法学和生物医学应用的 TDP-43 NTD 单体化策略。
