Department of Chemistry, Texas A&M University, College Station, Texas 77842, USA.
Biochemistry. 2011 Aug 23;50(33):7265-74. doi: 10.1021/bi200895k. Epub 2011 Aug 2.
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 k(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 k(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.
弗里德赖希共济失调（FRDA）是一种进行性神经退行性疾病，与蛋白 frataxin（FXN）的功能丧失有关，这种蛋白的功能丧失是由于 FXN 水平低，这是由于 GAA 三核苷酸重复扩展，或者偶尔由于 FXN 基因中的错义突变。在这里，研究人员确定了 FXN 变体的生化和结构特性，包括三种 FRDA 错义突变（N146K、Q148R 和 R165C）和三种相关突变体（N146A、Q148G 和 Q153A），以努力了解功能丧失的结构基础。体外测定表明，尽管这三种 FRDA 错义突变表现出相似的半胱氨酸脱硫酶和 Fe-S 簇组装活性丧失，但这些激活缺陷的原因是不同的。R165C 变体表现出比天然 FXN 更高的 k(cat)/K(M)，但与 NFS1、ISD11 和 ISCU2（SDU）复合物的结合较弱，而 Q148R 变体表现出六种测试的 FXN 变体中最低的 k(cat)/K(M)，并且仅表现出适度的结合缺陷。FXN 对 SDU Fe-S 组装复合物的结合亲和力顺序如下：FXN > Q148R > N146A > Q148G > N146K > Q153A > R165C。根据它们的生化特性，鉴定了四种不同类别的 FXN 变体。这些结构功能研究共同揭示了 Fe-S 组装复合物的结合和变构激活的决定因素，并深入了解了 FRDA 错义突变如何在功能上受到损害。
