Faraj Santiago E, Roman Ernesto A, Aran Martin, Gallo Mariana, Santos Javier
Instituto de Química y Físico-Química Biológicas, Universidad de Buenos Aires, Argentina.
FEBS J. 2014 Aug;281(15):3397-419. doi: 10.1111/febs.12869. Epub 2014 Jul 1.
Friedreich's ataxia (FRDA) is linked to a deficiency of frataxin (FXN), a mitochondrial protein involved in iron-sulfur cluster synthesis. FXN is a small protein with an α/β fold followed by the C-terminal region (CTR) with a nonperiodic structure that packs against the protein core. In the present study, we explored the impact of the alteration of the CTR on the stability and dynamics of FXN. We analyzed several pathological and rationally designed CTR mutants using complementary spectroscopic and biophysical approaches. The pathological mutation L198R yields a global destabilization of the structure correlating with a significant and highly localized alteration of dynamics, mainly involving residues that are in contact with L198 in wild-type FXN. Variant FXN 90-195, which is closely related to the FRDA-associated mutant FXN 81-193, conserves a globular shape with a native-like structure. However, the truncation of the CTR results in an extreme alteration of global stability and protein dynamics over a vast range of timescales and encompassing regions far from the CTR, as shown by proton-water exchange rates and (15) N-relaxation measurements. Increased sensitivity to proteolysis, observed in vitro for both mutants, suggests a faster degradation rate in vivo, whereas the enhanced tendency to aggregate exhibited by the truncated variant may account for the loss of functional FXN, with both phenomena providing an explanation as to why the alteration of the CTR causes FRDA. These results contribute to understanding how stability and activity are linked to protein motions and they might be useful for the design of target-specific ligands to control local protein motions for stability enhancement.
弗里德赖希共济失调（FRDA）与铁硫簇合成相关的线粒体蛋白frataxin（FXN）缺乏有关。FXN是一种小蛋白，具有α/β折叠结构，随后是C端区域（CTR），其具有非周期性结构，与蛋白质核心紧密堆积。在本研究中，我们探讨了CTR改变对FXN稳定性和动力学的影响。我们使用互补的光谱和生物物理方法分析了几种病理性和合理设计的CTR突变体。病理性突变L198R导致结构整体不稳定，这与动力学的显著且高度局部性改变相关，主要涉及野生型FXN中与L198接触的残基。与FRDA相关突变体FXN 81 - 193密切相关的变体FXN 90 - 195保留了类似天然结构的球状形状。然而，CTR的截断导致在广泛的时间尺度上以及远离CTR的区域内，整体稳定性和蛋白质动力学发生极端改变，这通过质子 - 水交换率和（15）N弛豫测量得以证明。在体外对这两种突变体均观察到对蛋白水解的敏感性增加，这表明其在体内的降解速度更快，而截断变体表现出的增强的聚集倾向可能解释了功能性FXN的丧失，这两种现象都解释了为什么CTR的改变会导致FRDA。这些结果有助于理解稳定性和活性如何与蛋白质运动相关联，并且它们可能有助于设计靶向特异性配体以控制局部蛋白质运动来增强稳定性。
