Takeda Agnes A S, Freitas Fernanda Z, Magro Angelo J, Bernardes Natalia E, Fernandes Carlos A H, Gonçalves Rodrigo D, Bertolini Maria Célia, Fontes Marcos R M
Departamento de Bioquimica e Tecnologia Quimica, Instituto de Biociências, Universidade Estadual Paulista, Botucatu, SP, Brazil.
Protein Pept Lett. 2013 Jan;20(1):8-16.
Neurospora crassa has been widely used as a model organism and contributed to the development of biochemistry and molecular biology by allowing the identification of many metabolic pathways and mechanisms responsible for gene regulation. Nuclear proteins are synthesized in the cytoplasm and need to be translocated to the nucleus to exert their functions which the importin-α receptor has a key role for the classical nuclear import pathway. In an attempt to get structural information of the nuclear transport process in N. crassa, we present herein the cloning, expression, purification and structural studies with N-terminally truncated IMPα from N. crassa (IMPα-Nc). Circular dichroism analysis revealed that the IMPα-Nc obtained is correctly folded and presents a high structural conservation compared to other importins-α. Dynamic light scattering, analytical size-exclusion chromatography experiments and molecular dynamics simulations indicated that the IMPα-Nc unbound to any ligand may present low stability in solution. The IMPα-Nc theoretical model displayed high similarity of its inner concave surface, which binds the cargo proteins containing the nuclear localization sequences, among IMPα from different species. However, the presence of non-conserved amino acids relatively close to the NLS binding region may influence the binding specificity of IMPα-Nc to cargo proteins.
粗糙脉孢菌已被广泛用作模式生物，通过鉴定许多负责基因调控的代谢途径和机制，为生物化学和分子生物学的发展做出了贡献。核蛋白在细胞质中合成，需要转运到细胞核才能发挥其功能，其中输入蛋白-α受体在经典的核输入途径中起关键作用。为了获得粗糙脉孢菌核运输过程的结构信息，我们在此展示了对粗糙脉孢菌N端截短的IMPα（IMPα-Nc）的克隆、表达、纯化及结构研究。圆二色性分析表明，所获得的IMPα-Nc折叠正确，与其他输入蛋白-α相比具有高度的结构保守性。动态光散射、分析型尺寸排阻色谱实验和分子动力学模拟表明，未与任何配体结合的IMPα-Nc在溶液中可能具有较低的稳定性。IMPα-Nc的理论模型显示，其与含有核定位序列的货物蛋白结合的内凹表面在不同物种的IMPα中具有高度相似性。然而，相对靠近核定位信号结合区域的非保守氨基酸的存在可能会影响IMPα-Nc与货物蛋白的结合特异性。
