Structure-Function Analysis of Friedreich's Ataxia Mutants Reveals Determinants of Frataxin Binding and Activation of the Fe-S Assembly Complex
Structure-Function Analysis of Friedreich's Ataxia Mutants Reveals Determinants of Frataxin Binding and Activation of the Fe-S Assembly Complex
复制标题
DOI：
10.1021/bi200895k
复制
发表时间：
2011-08-23
期刊：
影响因子：
2.9
通讯作者：
Barondeau, David P.
中科院分区：
文献类型：
--
作者：
Bridwell-Rabb, Jennifer;Winn, Andrew M.;Barondeau, David P.
Friedreich‘s共济失调(FRDA)是一种进行性神经退行性疾病，与蛋白质Frataxin(FXN)功能丧失有关，FXN是由于GAA三联体重复扩增导致FXN水平低下或偶尔FXN基因错义突变引起的。在这里，我们测定了FXN变异体的生化和结构特性，包括三个FRDA错义突变(N146K、Q148R和R165C)和三个相关突变体(N146A、Q148G和Q153A)，以努力了解功能丧失的结构基础。体外分析表明，尽管三个FRDA错义突变表现出相似的半胱氨酸脱硫酶和Fe-S簇组装活性的丧失，但这些激活缺陷的原因是不同的。R165C变异体的kappa(CAT)/K-M高于天然FXN，但与NFS1、ISD11和ISCU2(SDU)复合体的结合较弱，而Q148R变异体在所测试的六个FXN变异体中表现出最低的kappa(CAT)/K-M，仅有轻微的结合缺陷。SDU Fe S组装络合物的FXN结合亲和力顺序为：FXN>；Q148R>；N146A>；Q148G>；N146K>；Q153A>；R165C。根据FXN的生化特性，我们鉴定了四类不同的FXN变异体。总之，这些结构功能研究揭示了Fe-S组装复合体的结合和变构激活的决定因素，并为FRDA错义突变是如何在功能上受到损害提供了深入的见解。
Friedreich's ataxia (FRDA) is a progressive neurodegenerative disease associated with the loss of function of the protein frataxin (FXN) that results from low FXN levels due to a GAA triplet repeat expansion or, occasionally, from missense mutations in the FXN gene. Here biochemical and structural properties of FXN variants, including three FRDA missense mutations (N146K, Q148R, and R165C) and three related mutants (N146A, Q148G, and Q153A), were determined in an effort to understand the structural basis for the loss of function. In vitro assays revealed that although the three FRDA missense mutations exhibited similar losses of cysteine desulfurase and Fe-S cluster assembly activities, the causes for these activation defects were distinct. The R165C variant exhibited a kappa(cat)/K-M higher than that of native FXN but weak binding to the NFS1, ISD11, and ISCU2 (SDU) complex, whereas the Q148R variant exhibited the lowest kappa(cat)/K-M of the six tested FXN variants and only a modest binding deficiency. The order of the FXN binding affinities for the SDU Fe S assembly complex was as follows: FXN > Q148R > N146A > Q148G > N146K > Q153A > R165C. Four different classes of FXN variants were identified on the basis of their biochemical properties. Together, these structure-function studies reveal determinants for the binding and allosteric activation of the Fe-S assembly complex and provide insight into how FRDA missense mutations are functionally compromised.