Structural Basis of the Iron Storage Function of Frataxin from Single-Particle Reconstruction of the Iron-Loaded Oligomer † (original) (raw)
Related papers
Structure, 2006
Defects in the mitochondrial protein frataxin are responsible for Friedreich ataxia, a neurodegenerative and cardiac disease that affects 1:40,000 children. Here, we present the crystal structures of the ironfree and iron-loaded frataxin trimers, and a singleparticle electron microscopy reconstruction of a 24 subunit oligomer. The structures reveal fundamental aspects of the frataxin mechanism. The trimer has a central channel in which one atom of iron binds. Two conformations of the channel with different metalbinding affinities suggest that a gating mechanism controls whether the bound iron is delivered to other proteins or transferred to detoxification sites. The trimer constitutes the basic structural unit of the 24 subunit oligomer. The architecture of this oligomer and several features of the trimer structure demonstrate striking similarities to the iron-storage protein ferritin. The data reveal how stepwise assembly provides frataxin with the structural flexibility to perform two functions: metal delivery and detoxification.
Structure of Frataxin Iron Cores: An X-ray Absorption Spectroscopic Study †
Biochemistry, 2003
X-ray absorption spectroscopy at the iron K-edge indicates that the iron cores of human and yeast frataxin polymers assembled in Vitro are identical to each other and are similar but not identical to ferritin cores. Both frataxin polymers contain ferrihydrite, a biomineral composed of ferric oxide/hydroxide octahedra. The ferrihydrite in frataxin is less ordered than iron cores of horse spleen ferritin, having fewer face-sharing Fe-Fe interactions but similar double corner-sharing interactions. The extended X-ray absorption fine structure (EXAFS) analysis agrees with previous electron microscopy data showing that frataxin cores are composed of very small ferrihydrite crystallites.
Localized Unfolding at the Junction of Three Ferritin Subunits. A MECHANISM FOR IRON RELEASE?
Journal of Biological Chemistry, 1998
How and where iron exits from ferritin for cellular use is unknown. Twenty-four protein subunits create a cavity in ferritin where iron is concentrated >10 11-fold as a mineral. Proline substitution for conserved leucine 134 (L134P) allowed normal assembly but increased iron exit rates. X-ray crystallography of H-L134P ferritin revealed localized unfolding at the 3-fold axis, also iron entry sites, consistent with shared use sites for iron exit and entry. The junction of three ferritin subunits appears to be a dynamic aperture with a "shutter" that cytoplasmic factors might open or close to regulate iron release in vivo. Ferritins are vesicle-like assemblies of 24 polypeptide (4helix bundle) subunits that concentrate iron in cells by directing the formation of a ferric mineral in the hollow protein interior (8 nm diameter) (1-3). Effective cellular iron concentrations Ͼ10 11 times the solubility of the ferric ion are achieved by ferritins, which are found in microorganisms, plants, and animals. The complexity and the sophistication of the genetic regulation of the ferritins, involving both DNA and mRNA (4-7), emphasize the central role of iron and ferritin in life. Rates of Fe(II) oxidation, translocation of Fe(II) and Fe(III) (1.0-2.0 nm), and mineralization are all controlled by the protein (1, 2). Fe(II) release from ferritin following reduction of the mineral is slow and poorly understood (8, 9) but is important for the biosynthesis of iron-proteins, such as those required in respiration, photosynthesis, nitrogen fixation, and cell division, (1, 2) and as dietary iron (10). How and where the iron exits from ferritin in vivo is not known. We now show that localized unfolding in the assembled protein, at sites of cooperative subunit interactions, can increase
Drosophila Frataxin: An Iron Chaperone during Cellular Fe−S Cluster Bioassembly
Biochemistry, 2008
Frataxin, a mitochondrial protein that is directly involved in regulating cellular iron homeostasis, has been suggested to serve as an iron chaperone during cellular Fe-S cluster biosynthesis. In humans, decreased amounts or impaired function of frataxin causes the autosomal recessive neurodegenerative disorder Friedreich's ataxia. Cellular production of Fe-S clusters is accomplished by the Fe cofactor assembly platform enzymes Isu (eukaryotes) and IscU (prokaryotes). In this report, we have characterized the overall stability and iron binding properties of the Drosophila frataxin homologue (Dfh). Dfh is highly folded with secondary structural elements consistent with the structurally characterized frataxin orthologs. While the melting temperature (T M ≈ 59 °C) and chemical stability ([urea] 50% ≈ 2.4 M) of Drosophila frataxin, measured using circular dichroism (CD) and fluorescence spectroscopy, closely match values determined for the human ortholog, pure Dfh is more stable against autodegradation than both the human and yeast proteins. The ferrous iron binding affinity (K d ≈ 6.0 μM) and optimal metal to protein stoichiometry (1:1) for Dfh have been measured using isothermal titration calorimetry (ITC). Under anaerobic conditions with salt present, holo-Dfh is a stable iron-loaded protein monomer. Frataxin prevents reactive oxygen species-induced oxidative damage to DNA when presented with both Fe(II) and H 2 O 2. Ferrous iron bound to Dfh is high-spin and held in a partially symmetric Fe-(O/N) 6 coordination environment, as determined by X-ray absorption spectroscopy (XAS). Extended X-ray absorption fine structure (EXAFS) simulations indicate the average Fe-O/N bond length in Dfh is 2.13 Å, consistent with a ligand geometry constructed by water and carboxylate oxygens most likely supplied in part by surfaceexposed conserved acidic residues located on helix 1 and strand 1 in the structurally characterized frataxin orthologs. The iron-dependent binding affinity (K d ≈ 0.21 μM) and optimal holo-Dfh to Isu monomer stoichiometry (1:1) have also been determined using ITC. Finally, frataxin mediates the delivery of Fe(II) to Isu, promoting Fe-S cluster assembly in vitro. The Dfh-assisted assembly of Fe-S clusters occurs with an observed kinetic rate constant (k obs) of 0.096 min −1 .
Reconstituted and native iron-cores of bacterioferritin and ferritin
Journal of Molecular Biology, 1987
The structural and magnetic properties of the iron-cores of reconstituted horse spleen ferritin and Azotobacter vinelandii bacterioferritin have been investigated by high-resolution transmission electron microscopy, electron diffraction and Mossbauer spectroscopy. The structural properties of native horse spleen ferritin, native AZ. vinelandii, and native and reconstituted Pseudomonas aeruginosa bacterioferritins have also been determined. Reconstitution in the absence of inorganic phosphate at pH 7.0 showed sigmoidal behaviour in each protein but was approximately 30% faster in initial rate for the AZ. vinelandii protein when compared with horse spleen apoferritin. The presence of Zn'+ reduced the initial rate of Fe(I1) oxidation in AZ. vinelandii to 22% of the control rate. The iron-cores of the reconstituted bacterioferritins adopt defect ferrihydrite structures and are more highly ordered than their native counterparts, which are both amorphous, However, the blocking temperature for reconstituted AZ. vinelandii (22.2 K) is almost identical to that for the native protein (20 K). Particle size measurements indicate that, the reconstituted AZ. vinelandii cores are smaller in median diameter than the native cores and this reduction in particle volume (V) offsets the increased magnetocrystalline contribution to the magnetic anisotropy constant (K) in such a way that the magnetic anisotropy barrier (K V), and hence the blocking temperature, is similar for both proteins. Reconstituted horse spleen ferritin exhibits a similar blocking temperature (38 K) to that determined for the native protein, although it is structurally more disordered. The possibility of introducing structural and compositional modifications in both horse ferritin and bacterioferritins by in-vitro reconstitution suggests that these proteins do not function primarily as a crystallochemical-specific interface for core development in viva.
Journal of Molecular Biology, 2011
Frataxin is a mitochondrial protein with a central role in iron homeostasis. Defects in frataxin function lead to Friedreich's ataxia, a progressive neurodegenerative disease with childhood onset. The function of frataxin has been shown to be closely associated with its ability to form oligomeric species; however, the factors controlling oligomerization and the types of oligomers present in solution are a matter of debate. Using small-angle Xray scattering, we found that Co 2+ , glycerol, and a single amino acid substitution at the N-terminus, Y73A, facilitate oligomerization of yeast frataxin, resulting in a dynamic equilibrium between monomers, dimers, trimers, hexamers, and higher-order oligomers. Using X-ray crystallography, we found that Co 2+ binds inside the channel at the 3-fold axis of the trimer, which suggests that the metal has an oligomer-stabilizing role. The results reveal the types of oligomers present in solution and support our earlier suggestions that the trimer is the main building block of yeast frataxin oligomers. They also indicate that different mechanisms may control oligomer stability and oligomerization in vivo.
Journal of Biological Chemistry, 2013
Background: Iron-induced oligomerization of frataxin is still poorly understood. Results: The molecular basis of iron-induced oligomerization of yeast and bacterial frataxin is revealed. Catalyzed ferroxidation is required for correct oligomerization of Yfh1. Conclusion: Frataxin forms different oligomeric species at physiological conditions. Significance: Iron availability controls frataxin oligomerization, which in turn may control the processes that require iron delivery by frataxin.
Journal of the American Chemical Society
Biomineralization is mediated by specialized proteins that guide and control mineral sedimentation. In many cases, the active regions of these biomineralization proteins are intrinsically disordered. High-resolution structures of these proteins while they interact with minerals are essential for understanding biomineralization processes and the function of intrinsically disordered proteins (IDPs). Here we used the cavity of ferritin as a nanoreactor where the interaction between M6A, an intrinsically disordered iron-binding domain, and an iron oxide particle was visualized at high resolution by cryo-EM. Taking advantage of the differences in the electron-dose sensitivity of the protein and the iron oxide particles, we developed a method to determine the irregular shape of the particles found in our density maps. We found that the folding of M6A correlates with the detection of mineral particles in its vicinity. M6A interacts with the iron oxide particles through its C-terminal side, resulting in the stabilization of a helix at its N-terminal side. The stabilization of the helix at a region that is not in direct contact with the iron oxide particle demonstrates the ability of IDPs to respond to signals from their surroundings by conformational changes. These findings provide the first glimpse toward the long-suspected mechanism for biomineralization protein control over mineral microstructure, where unstructured regions of these proteins become more ordered in response to their interaction with the nascent mineral particles.
Human Molecular Genetics, 2002
Friedreich's ataxia (FRDA), an autosomal recessive cardio-and neurodegenerative disease, is caused by low expression of frataxin, a small mitochondrial protein, encoded in the nucleus. At the biochemical level, the lack of frataxin leads to dysregulation of mitochondrial iron homeostasis and oxidative damage, which eventually causes neuronal death. It is, however, still unclear whether frataxin is directly involved in iron binding, since the yeast orthologue, but not the human protein, has been shown to form large aggregates in the presence of large iron excess. We have compared the properties of three proteins from the frataxin family-the bacterial CyaY from Escherichia coli, the yeast Yfh1 and human frataxin-as representative of organisms of increasing complexity. We show that the three proteins have the same fold but different thermal stabilities and iron-binding properties. While human frataxin has no tendency to bind iron, CyaY forms ironpromoted aggregates with a behaviour similar to that of yeast frataxin. However, aggregation can be competed by chelator agents or by ionic strength. At physiological salt conditions, almost no aggregation is observed. The design of mutants produced to identify the protein surface involved in iron-promoted aggregation allows us to demonstrate that the process is mediated by a negatively charged surface ridge. Mutation of three of these residues is sufficient to convert CyaY in a protein with properties similar to those of human frataxin. On the other hand, mutation of the exposed surface of the b sheet, which contains most of the conserved residues, does not affect aggregation, suggesting that iron binding is a non-conserved part of a more complex cellular function of frataxins.