iBB- Institute for Bioengineering and Bioscience, Instituto Superior Técnico, Universidade de Lisboa, 1049-001, Lisboa, Portugal.
IDIBE, Universidad Miguel Hernández, 03202 Elche, Alicante, Spain.
Biochim Biophys Acta Gen Subj. 2020 Jul;1864(7):129609. doi: 10.1016/j.bbagen.2020.129609. Epub 2020 Mar 29.
Eukaryotic cells have a continuous transit of macromolecules between the cytoplasm and the nucleus. Several carrier proteins are involved in this transport. One of them is importin α, which must form a complex with importin β to accomplish its function, by domain-swapping its 60-residue-long N terminus. There are several human isoforms of importin α; among them, importin α3 has a particularly high flexibility.
We studied the conformational stability of intact importin α3 (Impα3) and its truncated form, where the 64-residue-long, N-terminal importin-β-binding domain (IBB) has been removed (ΔImpα3), in a wide pH range, with several spectroscopic, biophysical, biochemical methods and with molecular dynamics (MD).
Both species acquired native-like structure between pH 7 and 10.0, where Impα3 was a dimer (with an apparent self-association constant of ~10 μM) and ΔImpα3 had a higher tendency to self-associate than the intact species. The acquisition of secondary, tertiary and quaternary structure, and the burial of hydrophobic patches, occurred concomitantly. Both proteins unfolded irreversibly at physiological pH, by using either temperature or chemical denaturants, through several partially folded intermediates. The MD simulations support the presence of these intermediates.
The thermal stability of Impα3 at physiological pH was very low, but was higher than that of ΔImpα3. Both proteins were stable in a narrow pH range, and they unfolded at physiological pH populating several intermediate species.
The low conformational stability explains the flexibility of Impα3, which is needed to carry out its recognition of complex cargo sequences.
真核细胞的细胞质和细胞核之间有大分子的持续转运。有几种载体蛋白参与这种运输。其中一种是导入蛋白α（importin α），它必须与导入蛋白β形成复合物，通过其 60 个残基长的 N 端的结构域交换来完成其功能。导入蛋白α有几种人类同工型；其中，导入蛋白α3 的灵活性特别高。
我们在较宽的 pH 范围内，使用几种光谱学、生物物理学、生物化学方法和分子动力学（MD）研究了完整的导入蛋白α3（Impα3）及其截断形式（去除了 64 个残基长的 N 端导入蛋白-β结合域（IBB）的 ΔImpα3）的构象稳定性。
两种物质在 pH 7 到 10.0 之间都获得了类似天然的结构。在这个 pH 范围内，Impα3 是二聚体（具有约 10 μM 的表观自缔合常数），而 ΔImpα3 比完整的物质更倾向于自缔合。二级、三级和四级结构的获得以及疏水性斑块的埋藏是同时发生的。两种蛋白质在生理 pH 下通过使用温度或化学变性剂不可逆地展开，通过几种部分折叠的中间产物。MD 模拟支持这些中间产物的存在。
Impα3 在生理 pH 下的热稳定性非常低，但高于 ΔImpα3。在较窄的 pH 范围内，两种蛋白质都很稳定，它们在生理 pH 下展开，形成几种中间产物。
低构象稳定性解释了 Impα3 的灵活性，这是其识别复杂货物序列所必需的。
