Fig 1.
SAXS measurement of human frataxin FXN81-210.
A) Small-angle X-ray scattering profile of FXN81-210. 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 FXN81-210 (blue) plotted against iron-to-protein ratio.
Fig 2.
DLS studies of iron-dependent oligomerization of human frataxin FXN81-210.
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 FXN81-210 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.
Table 1.
Average size and distribution of particles of FXN81-210 obtained from DLS measurements and the effect of iron chelators.
Table 2.
The effect of ferric iron on the oligomerization of human frataxin FXN81-210.
Table 3.
Average size and distribution of particles of CyaY obtained from DLS measurements and the effect of iron chelators.
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 FXN81-210 incubated with iron.
A) FXN81-210 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 FXN81-210, while the second peak corresponds to oligomeric protein in the tetrameric to hexameric size range of 50–80 kDa, B) FXN81-210 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 FXN81-210, can be seen. For the sample with BIPY a main peak is at the size of monomeric FXN81-210, while the second broader peak is at the size of oligomers in the tetrameric-hexameric range of 50–80 kDa C) FXN81-210 incubated with 6:1 molar ratio of iron-to-protein and 3x excess DFO (blue). The main peak, corresponds to monomeric FXN81-210, 2 small peaks correspond to approximately ~75 kDa and 440 kDa.
Fig 5.
Negative staining TEM images of iron-induced FXN81-210 oligomers.
A) Monomeric FXN81-210. B) FXN81-210 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) FXN81-210 incubated with iron at 6:1 molar ratio of iron-to-protein and with deferiprone added. D) FXN81-210 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 FXN81-210 oligomers.
A) SDS-PAGE gel of crosslinked FXN81-210 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.