Structure–Function Analysis of Friedreich’s Ataxia Mutants Reveals Determinants of Frataxin Binding and Activation of the Fe–S Assembly Complex

Abstract

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 kcat/KM higher than that of native FXN but weak binding to the NFS1, ISD11, and ISCU2 (SDU) complex, whereas the Q148R variant exhibited the lowest kcat/KM 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.

Selected missense mutations impair frataxin processing in Friedreich ataxia

“…The inability to detect FXN L106S and FXN G137V by immunostaining and western blot supports modeling studies suggesting that mutations residing within the protein core decrease protein stability 9. In vitro functional studies have also characterized FXN R165C and FXN W155R as dysfunctional mutations, causing decreased binding of FXN to Fe‐S cluster assembly complex 16, 17. Moreover, FXN R165C and FXN W155R had levels of FXN 81–210 that were comparable to FXN WT , and there was no evidence for impaired processing of these two mutant forms from FXN 42–210 to FXN 81–210 .…”

“…In contrast, R165C, W155R, G130V, and I154F mutations are suspected to produce stable protein. However, R165C and W155R lead to biochemical deficiencies in vitro 16, 17. The mechanism behind the disease‐causing features of G130V and I154F is less clear, having been suggested to reflect abnormal maturation or dysfunctional FXN in different models 9.…”

Missense Mutations Linked to Friedreich Ataxia Have Different but Synergistic Effects on Mitochondrial Frataxin Isoforms

“…As individuals carrying one GAA expansion were phenotypically normal, whereas compound heterozygotes for one GAA expansion and one missense mutation were affected with Friedreich ataxia, it was established early on that single missense mutations in FRDA could have severe clinical consequences (10); however, the underlying mechanisms have thus far remained elusive. Two of the most aggressive missense mutations, I154F and W155R, were previously characterized in the context of the FXN 81-210 isoform or a shorter variant, FXN 91-210 (19,(25)(26)(27). Here, we have compared the effects of the I154F and W155R mutations on the two most abundant mitochondrial FXN isoforms, FXN and FXN .…”

“…1A). It has been proposed that Trp-155 creates a binding site on the surface of the FXN 81-210 monomer for the NFS1-ISD11-ISCU complex and that this site is disrupted directly by the W155R mutation and indirectly by the I154F mutation via secondary effects on Trp-155 and neighboring residues (26,27). On the other hand, the crystal structure of the yeast frataxin trimer shows that this highly conserved residue (Trp-131 in the yeast protein) mediates direct contacts between the ␤ sheet of one subunit and the N-terminal region of a neighboring subunit (33,34) (Fig.…”

“…To date, however, the functional consequences of such mutations have been studied almost exclusively in the context of FXN 81-210 (19,(25)(26)(27)). Here we show that two of the most clinically severe mutations, I154F and W155R, have multiple distinct effects on the biogenesis, stability, and functions of FXN and FXN .…”