Iron-induced oligomerization of human FXN81-210 and bacterial CyaY frataxin and the effect of iron chelators

Abstract

Patients suffering from the progressive neurodegenerative disease Friedreich's ataxia have reduced expression levels of the protein frataxin. Three major isoforms of human frataxin have been identified, FXN42-210, FXN56-210 and FXN81-210, of which FXN81-210 is considered to be the mature form. Both long forms, FXN42-210 and FXN56-210, have been shown to spontaneously form oligomeric particles stabilized by the extended N-terminal sequence. The short variant FXN81-210, on other hand, has only been observed in the monomeric state. However, a highly homologous E. coli frataxin CyaY, which also lacks an N-terminal extension, has been shown to oligomerize in the presence of iron. To explore the mechanisms of stabilization of short variant frataxin oligomers we compare here the effect of iron on the oligomerization of CyaY and FXN81-210. Using dynamic light scattering, small-angle X-ray scattering, electron microscopy (EM) and cross linking mass spectrometry (MS), we show that at aerobic conditions in the presence of iron both FXN81-210 and CyaY form oligomers. However, while CyaY oligomers are stable over time, FXN81-210 oligomers are unstable and dissociate into monomers after about 24 h. EM and MS studies suggest that within the oligomers FXN81-210 and CyaY monomers are packed in a head-to-tail fashion in ring-shaped structures with potential iron-binding sites located at the interface between monomers. The higher stability of CyaY oligomers can be explained by a higher number of acidic residues at the interface between monomers, which may result in a more stable iron binding. We also show that CyaY oligomers may be dissociated by ferric iron chelators deferiprone and DFO, as well as by the ferrous iron chelator BIPY. Surprisingly, deferiprone and DFO stimulate FXN81-210 oligomerization, while BIPY does not show any effect on oligomerization in this case. The results suggest that FXN81-210 oligomerization is primarily driven by ferric iron, while both ferric and ferrous iron participate in CyaY oligomer stabilization. Analysis of the amino acid sequences of bacterial and eukaryotic frataxins suggests that variations in the position of the acidic residues in helix 1, β-strand 1 and the loop between them may control the mode of frataxin oligomerization.

Fig 1. SAXS measurement of human frataxin FXN^81-210.

A) Small-angle X-ray scattering profile of FXN^81-210. The experimental data are shown as circles. B) Pair-distribution function calculated using GNOM and the SAXS profile. C) Green spheres represent an ab initio model calculated using GASBOR, and superimposed is the X-ray crystallographic model of human frataxin (orange cartoon) residues 90–210 (PDB ID: 1EKG). The nine missing residues (orange spheres) were modeled using CORAL [42]. D) Porod volume for Yfh1 (black), CyaY (red), and FXN^81-210 (blue) plotted against iron-to-protein ratio.

Fig 2. DLS studies of iron-dependent oligomerization of human frataxin FXN^81-210.

A) Measurements after 30 min of incubation with iron at 2:1 equivalents of iron-to-protein (magenta), 4:1 (green), and 10:1 (blue). B) Measurements after 60 min of incubation showing buildup of oligomers. In black is monomeric human FXN^81-210 without the addition of iron. On the x-axis is the hydrodynamic radius of the particles and on the y-axis is the volume percentage of particles.

Fig 3. DLS studies of iron-dependent oligomerization of E. coli CyaY.

A) CyaY was incubated for 1 h at 2:1 iron-to-protein ratio (green); 6:1, magenta; 10:1, blue. Dashed lines show the same sample after 26 h of incubation. In black, monomeric CyaY without addition of iron. On the x-axis the hydrodynamic radius and on the y-axis the volume-percentage of particles are shown. B) CyaY oligomerization with and without DFO. The protein was incubated at 6:1 (red) and 8:1 (green) iron equivalents; in black the protein without iron addition; dashed line show the sample after the addition of DFO.

Fig 4. Size exclusion chromatography of human FXN^81-210 incubated with iron.

A) FXN^81-210 incubated with 10:1 molar ratio of iron-to-protein for 30 minutes (red) and 1 h (dark gray). The main peak corresponds to monomeric FXN^81-210, while the second peak corresponds to oligomeric protein in the tetrameric to hexameric size range of 50–80 kDa, B) FXN^81-210 incubated with 6:1 molar ratio of iron-to-protein and 3 x excess of BIPY (green) and deferiprone (black). For the sample with deferiprone only one peak, at the size of monomeric FXN^81-210, can be seen. For the sample with BIPY a main peak is at the size of monomeric FXN^81-210, while the second broader peak is at the size of oligomers in the tetrameric-hexameric range of 50–80 kDa C) FXN^81-210 incubated with 6:1 molar ratio of iron-to-protein and 3x excess DFO (blue). The main peak, corresponds to monomeric FXN^81-210, 2 small peaks correspond to approximately ~75 kDa and 440 kDa.

Fig 5. Negative staining TEM images of iron-induced FXN^81-210 oligomers.

A) Monomeric FXN^81-210. B) FXN^81-210 after 30 min of incubation with iron at 6:1 of iron-to-protein molar ratio. Ring-shaped particles are marked by black circles. C) FXN^81-210 incubated with iron at 6:1 molar ratio of iron-to-protein and with deferiprone added. D) FXN^81-210 incubated with iron at 6:1 molar ratio of iron-to-protein and with DFO added. Ring-shaped particles and larger spherical particles are marked by black circles. E) Class averages of the ring-shaped structures obtained from 1265 particles. The tetrameric character of the ring-structures can be clearly seen on some of the class averages. The EM images have a magnification of 55,000x.

Fig 6. Crosslinking of iron- and hydrogen peroxide-induced FXN^81-210 oligomers.

A) SDS-PAGE gel of crosslinked FXN^81-210 incubated with iron (II) at a 6:1 molar ratio of iron-to-protein and hydrogen peroxide with bands of monomeric, dimeric, trimeric, and tetrameric size indicated. B) Crystallographic structure of human frataxin (PDB entry 1EKG) was used here to generate a head-to-tail dimer. The regions of the structure shown in [33] to have reduced deuterium incorporation after Fe³⁺ binding are highlighted.

Fig 7. The interface between subunits in the head-to-tail arrangement.

A) Human frataxin interface with residues D112, D115, D122, and D124 marked in magenta. The N-terminus is colored in pink and the different monomers within the dimer are shown in yellow (head) and green (tail). A crystal structure (PDB entry 1EKG) was used for preparing the figure. B) CyaY interface and the residues making up the potential metal binding sites are shown. Residues H7, E19, D22, and D23, which may build up the first metal binding site, are shown as red sticks; D3, H58, and D25 may participate in the second site (yellow sticks); and H70, D29, and E44 (blue sticks) in the third. Gray spheres show Europium ions bound in the 2P1X crystal structure. Residues involved in metal binding in the crystal structures of CyaY in complex with Co and/or Eu are labeled. The different monomers in the dimer are shown in green (head) and blue (tail). Crystal structures (PDB entry 2P1X, 2EFF, and 1EW4) were used in the preparation of the figue.