Structure of the human frataxin-bound iron-sulfur cluster assembly complex provides insight into its activation mechanism.

The core machinery for de novo biosynthesis of iron-sulfur clusters (ISC), located in the mitochondria matrix, is a five-protein complex containing the cysteine desulfurase NFS1 that is activated by frataxin (FXN), scaffold protein ISCU, accessory protein ISD11, and acyl-carrier protein ACP. Deficiency in FXN leads to the loss-of-function neurodegenerative disorder Friedreich's ataxia (FRDA). Here the 3.2 Å resolution cryo-electron microscopy structure of the FXN-bound active human complex, containing two copies of the NFS1-ISD11-ACP-ISCU-FXN hetero-pentamer, delineates the interactions of FXN with other component proteins of the complex. FXN binds at the interface of two NFS1 and one ISCU subunits, modifying the local environment of a bound zinc ion that would otherwise inhibit NFS1 activity in complexes without FXN. Our structure reveals how FXN facilitates ISC production through stabilizing key loop conformations of NFS1 and ISCU at the protein-protein interfaces, and suggests how FRDA clinical mutations affect complex formation and FXN activation.

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

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, ß-strand 1 and the loop between them may control the mode of frataxin oligomerization.

Negative staining across holes: application to fibril and tubular structures.

The negative staining technique, when used with holey carbon support films, presents superior imaging conditions than is the case when samples are adsorbed to continuous carbon films. A demonstration of this negative staining approach is presented, using ammonium molybdate in combination with trehalose, applied to several fibrillar and tubular samples. Fibrils formed from the amyloid-beta peptide and the protease inhibitor pepstain A spread very well unsupported across holes and the different polymorphic fibril forms can be readily assessed. However, tubular forms of amyloid-beta have a tendency to be flattened, due to surface tension forces prior to and during specimen drying. Sub-fibril assembly forms and D-banded rat tail type 1 collagen fibres are presented. The air-dried collagen images produced are shown to contain almost as much detail as those obtainable by cryo-negative staining. Fragile DNA and DNA-protein nanotubes are also shown to yield superior quality images to those produced on continuous carbon films. The iron-storage protein, frataxin, creates elongated oligomeric assemblies, containing bound ferrihydrite microcrystals. The iron particles within these flexuous oligomers can be defined in the presence of ammonium molybdate, but they are more readily demonstrated if the frataxin is spread across holes in the presence of trehalose alone. The samples used here serve to show the likely benefit obtainable from negative staining across holes for a range of other fibrillar and tubular samples in biology, medicine and nanobiotechnology.

Structural response of Photosystem 2 to iron deficiency: characterization of a new photosystem 2-IdiA complex from the cyanobacterium Thermosynechococcus elongatus BP-1.

Iron deficiency triggers various processes in cyanobacterial cells of which the synthesis of an additional antenna system (IsiA) around photosystem (PS) 1 is well documented [T.S. Bibby, J. Nield, J. Barber, Iron deficiency induces the formation of an antenna ring around trimeric photosystem I in cyanobacteria, Nature 412 (2001) 743-745, E.J. Boekema, A. Hifney, A.E. Yakushevska, M. Piotrowski, W. Keegstra, S. Berry, K.P. Michel, E.K. Pistorius, J. Kruip, A giant chlorophyll-protein complex induced by iron deficiency in cyanobacteria, Nature 412 (2001) 745-748]. Here we show that PS2 also undergoes prominent structural changes upon iron deficiency: Prerequisite is the isolation and purification of a PS2-IdiA complex which is exclusively synthesized under these conditions. Immunoblotting in combination with size exclusion chromatography shows that IdiA is only bound to dimeric PS2. Using single particle analysis of negatively stained specimens, IdiA can be localized in averaged electron micrographs on top of the CP43 subunit facing the cytoplasmic side in a model derived from the known 3D structure of PS2 [B. Loll, J. Kern, W. Saenger, A. Zouni, J. Biesiadka, Towards complete cofactor arrangement in the 3.0 A resolution structure of photosystem II, Nature 438 (2005) 1040-4]. The presence of IdiA as integral part of PS2 is the first example of a new PS2 protein being expressed under stress conditions, which is missing in highly purified PS2 complexes isolated from iron-sufficient cells.

A light and electron microscopic study of divalent metal transporter-1 distribution in the rat hippocampus, after kainate-induced neuronal injury.

An accumulation of iron occurs in the hippocampus of rats injected with kainate over time, but thus far whether this accumulation is associated with any changes in expression of iron transporters is not known. The present study was therefore carried out using an antibody to the divalent metal transporter-1 (DMT-1) and immunoblot and immunocytochemical analyses to elucidate possible changes in expression of the transporter in the rat hippocampus after kainate injections. A significant increase in density ratios of DMT-1/beta-actin bands was observed in Western blots in the 1-week, 1-month, and 2-months post-kainate-injected hippocampus, compared to uninjected and 1-day post-kainate-injected hippocampus. The increase in DMT-1 protein was paralleled by an increase in DMT-1 immunoreactivity in astrocytes. Light staining for DMT-1 was observed in the uninjected, saline-injected, and 1-day post-kainate-injected rat hippocampus. In contrast, an upregulation of DMT-1 was observed in reactive glial cells at 1 week, 1 month, and 2 months post-kainate injection. Electron microscopy confirmed that the glial cells had morphological features of astrocytes. DMT-1 is a cellular iron transporter responsible for transport of metal ions from the plasma membrane to endosomes. The observation that DMT-1 is present on astrocytic end feet in contact with blood vessels suggests that these cells may be involved in uptake of iron from endothelial cells.