SAXS and stability studies of iron-induced oligomers of bacterial frataxin CyaY

Abstract

Frataxin is a highly conserved protein found in both prokaryotes and eukaryotes. It is involved in several central functions in cells, which include iron delivery to biochemical processes, such as heme synthesis, assembly of iron-sulfur clusters (ISC), storage of surplus iron in conditions of iron overload, and repair of ISC in aconitase. Frataxin from different organisms has been shown to undergo iron-dependent oligomerization. At least two different classes of oligomers, with different modes of oligomer packing and stabilization, have been identified. Here, we continue our efforts to explore the factors that control the oligomerization of frataxin from different organisms, and focus on E. coli frataxin CyaY. Using small-angle X-ray scattering (SAXS), we show that higher iron-to-protein ratios lead to larger oligomeric species, and that oligomerization proceeds in a linear fashion as a results of iron oxidation. Native mass spectrometry and online size-exclusion chromatography combined with SAXS show that a dimer is the most common form of CyaY in the presence of iron at atmospheric conditions. Modeling of the dimer using the SAXS data confirms the earlier proposed head-to-tail packing arrangement of monomers. This packing mode brings several conserved acidic residues into close proximity to each other, creating an environment for metal ion binding and possibly even mineralization. Together with negative-stain electron microscopy, the experiments also show that trimers, tetramers, pentamers, and presumably higher-order oligomers may exist in solution. Nano-differential scanning fluorimetry shows that the oligomers have limited stability and may easily dissociate at elevated temperatures. The factors affecting the possible oligomerization mode are discussed.

Fig 1. Iron-dependent oligomerization of bacterial frataxin (CyaY).

A) SAXS scattering profiles for apo- and iron-induced CyaY oligomers at different iron-to-protein ratios. B) The double-logarithmic curve for the same SAXS scattering profiles showing apo-CyaY and the oligomeric mixtures of iron-incubated samples.

Fig 2. The TR-SAXS parameters for iron-induced CyaY oligomers.

A) Radius of gyration (R_g) and B) maximum distance (D_max) plotted against the time of incubation with iron for the sample with 7:1 iron-to-protein ratio.

Fig 3. SEC-SAXS for a 7:1 iron-to-CyaY ratio.

A) Elution profile (normalized absorbance at 280 nm) for the sample with 8 mg/ml CyaY concentration loaded on a Superdex 75 gel filtration column after the elimination of high oligomeric species through 100 kDa concentrators optimized for SEC-SAXS. B) SEC-SAXS results for the same sample at protein concentration of 56 mg/ml. Each frame corresponds to 1-second exposure time and is plotted against I_sum (blue) and R_g (red). Frames that showed higher stability in R_g for the first peak were averaged, and the buffer was subtracted for data processing. The second peak was for monomeric CyaY.

Fig 4. Rigid-body modeling for iron-induced CyaY dimer.

The crystallographic structure of monomeric CyaY (PDB entry: 1ew4) was used to generate the CyaY dimer structure using SASREF. Two models were obtained with A) head-to-tail and B) head-to-head arrangement of monomers. C) SASREF modeling fit of the head-to-tail dimer to the SEC-SAXS data. D) The head-to-tail model docked into the SAXS-filtered ab initio model with the corresponding P(r) plot shown in the insert.

Fig 5. OLIGOMER fitting for the 1:2 iron-to-protein ratio.

The experimental SAXS data are represented by circles, while the corresponding fit of OLIGOMER is shown as a red line. A) 1:2 iron-to-protein ratio (1 mg/ml of CyaY) and B) 1:2 iron-to-protein ratio (3 mg/ml CyaY) as performed in an earlier study [22].

Fig 6. Negatively stained TEM images of iron-induced CyaY oligomers.

A) & B) Iron-incubated sample of CyaY at 7:1 iron-to-protein ratio. Particles of different sizes can be seen in the images (55000x magnification).

Fig 7. Nano-DSF analysis of iron-induced CyaY oligomer samples.

A) The F_350nm/F_330nm is plotted against the temperature gradient. B) The first derivative for the F_350nm/F_330nm curve against the temperature gradient from which the T_m for each sample was derived.

Fig 8. Alignment of CyaY sequences.

The acidic residues in the sequence are boxed in red for clarity. CYAY_ECOLI–E. coli; CYAY_PSYIN—Psychromonas ingrahamii; CYAY_BURCJ—Burkholderia cenocepacia.

Fig 9. Burkholderia cenocepacia CyaY monomer assembly in the crystals (PDB entry: 4JPD).

A) The hexamer is built up by two trimers packed against each other around the threefold symmetry axis of the crystal. One trimer layer is shown in yellow and the other in red. The N- and C-termini of the subunits are labeled in blue. B) One of the trimer layers of the hexamer showing the arrangement of the monomers.

Fig 10. Helical wheel representation of helix 1 of five frataxin structure.

(A) Human frataxin; residues (94–114, PDB entry 1EKG) (B) Yeast frataxin Yfh1 (residues 76–88; PDB entry 3OEQ) (C) E. coli CyaY; (residues 3–22; PDB entry 1EW4) (D) Psychromonas ingrahamii CyaY (residues 3–24; PDB entry 4HS5) (E) Burkholderia cenocepacia CyaY (residues 4–26; PDB entry 4JPD). Acidic residues are shown as red filled circles, basic residues as blue, polar in magenta and hydrophobic in yellow. The letters represent the amino acids in one-letter code. Figure prepared using the HeliQuest server [60].