Oligomerization of human and bacterial frataxin : Structural and functional studies

Friedreich’s ataxia (FRDA) is an autosomal recessive neurodegenerative disease caused by defeciency in frataxin, a highly conserved protein central to iron homeostasis in mirochondria. The function of frataxin is related to its capability to bind, store and deliver iron to different biochemical process such as iron-sulfur cluster assembly and heme biosynthesis. Most FRDA pateints have extended GAA codon repeats in the first intron of the frataxin gene, which results in decreased protein expression. Low levels of frataxin lead to dysregulation of mitochondrial function, iron accumulation, increased levels of ROS production and subsequent mitochondrial protein and DNA damage.Two isoforms of human frataxin, FXN42-210 and FXN56-210, as well as yeast frataxin Yfh1 have been shown to build up oligomeric species stabilized by a long non-conserved N-terminal extension. On the other hand, the short form of human frataxin FXN81-210, which is also the most abundant isoform of the protein in cells, appeared to function only as a monomer. Within this thesis we study the oligomerization of the short form of human frataxin FXN81-210 , and compare it to the oligomerization of bacterial frataxin CyaY. The study is based on several experimental methods, including dynamic light scattering (DLS), small angle X-ray scattering (SAXS), electron microscopy (EM), crosslinking mass spectrometry (CXMS), X-ray absorption spectroscopy (XAS), and nano-differential scanning fluorimetry (nano-DSF).The results show that both FXN81-210 and CyaY are able to form oligomers in the presence of iron at aerobic conditions. However, while iron-induced CyaY oligomers had high stability over time, FXN81-210 oligomers dissociated into monomeric species after about 24 hours. Surprisingly, ferric iron chelators as well as hydrogen peroxide had the ability to enhance the formation of FXN81-210 oligomers. On the other hand, the presence of the same chelators resulted in the dissociation of higher order CyaY oligomeric into lower order oligomers and monomers. XAS studies indicated that large FXN81-210 oligomers are stabilized by a ferrihydrite mineral, which is presumably formed during oxidation of bound iron.CXMS, SAXS and EM were used in the analysis of the oligomeric structures. The data suggested that for both FXN81-210 and CyaY the iron induced larger oligomers are built up by dimers arranged in a head-to-tail mode. SAXS data were also used to study the effect of iron concentration relative to protein concentration on iron-induced CyaY oligomers, showing increased build up of oligomers with increasing iron concentration.Nano-DSF measurements on iron-induced CyaY oligomers were used for additional assessment of CyaY oligomer stabilty. The data showed two melting points a lower temperature point corresponding to oligomer dissociation and a high temperature point corresponding to monomer denaturation. This suggests that CyaY may build transient oligomers in cells in response to conditions of elevated iron content.