Towards a structural understanding of Friedreich’s ataxia: the solution structure of frataxin

Musco, Giovanna; Stier, Günter; Kolmerer, Bernhard; Adinolfi, Salvatore; Martin, Stephen R.; Frenkiel, Thomas A.; Gibson, Toby J.; Pastore, Annalisa

doi:10.1016/s0969-2126(00)00158-1

Cited by 156 publications

(176 citation statements)

References 44 publications

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“…FXN 90-210 exhibits a spectrum of excellent quality, which is very similar to the one reported previously [6]. Similarly, FXN L198R displays a good HSQC spectrum, with the expected number of peaks, all of them well dispersed and with uniform line widths.…”

Section: A B C Dsupporting

confidence: 82%

The alteration of the C‐terminal region of human frataxin distorts its structural dynamics and function

et al. 2014

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Friedreich's ataxia (FRDA) is linked to a deficiency of frataxin (FXN), a mitochondrial protein involved in iron-sulfur cluster synthesis. FXN is a small protein with an a/b fold followed by the C-terminal region (CTR) with a nonperiodic structure that packs against the protein core. In the present study, we explored the impact of the alteration of the CTR on the stability and dynamics of FXN. We analyzed several pathological and rationally designed CTR mutants using complementary spectroscopic and biophysical approaches. The pathological mutation L198R yields a global destabilization of the structure correlating with a significant and highly localized alteration of dynamics, mainly involving residues that are in contact with L198 in wild-type FXN. , which is closely related to the FRDA-associated mutant FXN 81-193, conserves a globular shape with a native-like structure. However, the truncation of the CTR results in an extreme alteration of global stability and protein dynamics over a vast range of timescales and encompassing regions far from the CTR, as shown by proton-water exchange rates and 15 N-relaxation measurements. Increased sensitivity to proteolysis, observed in vitro for both mutants, suggests a faster degradation rate in vivo, whereas the enhanced tendency to aggregate exhibited by the truncated variant may account for the loss of functional FXN, with both phenomena providing an explanation as to why the alteration of the CTR causes FRDA. These results contribute to understanding how stability and activity are linked to protein motions and they might be useful for the design of target-specific ligands to control local protein motions for stability enhancement.

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Section: A B C Dsupporting

confidence: 82%

The alteration of the C‐terminal region of human frataxin distorts its structural dynamics and function

et al. 2014

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“…7). Monomer is activated by Fe(II) in the presence of O 2 and forms an oligomer, ␣ 3 , with a negatively charged inner surface (63,64) that sequesters Fe(II) from the solution (Fig. 7A).…”

Section: Discussionmentioning

confidence: 99%

Yeast Frataxin Sequentially Chaperones and Stores Iron by Coupling Protein Assembly with Iron Oxidation

Park

Gakh

O'Neill

et al. 2003

Journal of Biological Chemistry

151

160

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We have investigated the mechanism of frataxin, a conserved mitochondrial protein involved in iron metabolism and neurodegenerative disease. Previous studies revealed that the yeast frataxin homologue (mYfh1p) is activated by Fe(II) in the presence of O 2 and assembles stepwise into a 48-subunit multimer (␣ 48 ) that sequesters >2000 atoms of iron in 2-4-nm cores structurally similar to ferritin iron cores. Here we show that mYfh1p assembly is driven by two sequential iron oxidation reactions: A ferroxidase reaction catalyzed by mYfh1p induces the first assembly step (␣ 3 ␣ 3 ), followed by a slower autoxidation reaction that promotes the assembly of higher order oligomers yielding ␣ 48 . Depending on the ionic environment, stepwise assembly is associated with accumulation of 50 -75 Fe(II)/subunit. Initially, this Fe(II) is loosely bound to mYfh1p and can be readily mobilized by chelators or made available to the mitochondrial enzyme ferrochelatase to synthesize heme. Transfer of mYfh1p-bound Fe(II) to ferrochelatase occurs in the presence of citrate, a physiologic ferrous iron chelator, suggesting that the transfer involves an intermolecular interaction. If mYfh1p-bound Fe(II) is not transferred to a ligand, iron oxidation, and mineralization proceed to completion, Fe(III) becomes progressively less accessible, and a stable iron-protein complex is formed. Iron oxidation-driven stepwise assembly is a novel mechanism by which yeast frataxin can function as an iron chaperone or an iron store.

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“…The proteins were purified as previously reported [11]; [12] ; [13]. In short, IscU and IscS were subcloned by PCR from bacterial genomic DNA and individually expressed from a pET-30 vector (EMBL Heidelberg) as fusion proteins with His-tagged glutathione-S-transferase (GST) and a cleavage site for Tobacco Etch Virus (TEV) protease which leaves, after cleavage, two additional amino acids (Gly-Ala) at the protein N-terminus.…”

Section: Materials and Methods Protein Productionmentioning

confidence: 99%

Of the vulnerability of orphan complex proteins: The case study of the E. coli IscU and IscS proteins

Prischi¹,

Pastore²,

Carroni³

et al. 2010

Protein Expression and Purification

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IscS and IscU, the two central protein components of the iron sulfur cluster assembly machinery, form a complex that is still relatively poorly characterized. In an attempt to standardize the purification of these proteins for structural studies we have developed a protocol to produce them individually in high concentration and purity. We show that IscS is a rather robust protein as long as it is produced in a PLP loaded form and that this co-factor is essential for fold stability and enzyme activity. In contrast to previous evidence, we also propose that, in contrast with previous evidence, IscU is a thermodynamically stable protein with a well defined fold but, when produced in isolation, is a 'complex-orphan protein' that is prone to unfolding if not stabilised by a co-factor or a protein partner. Our work will facilitate further structural and functional studies of these proteins and eventually lead to a better understanding of the whole machinery.

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Towards a structural understanding of Friedreich’s ataxia: the solution structure of frataxin

Cited by 156 publications

References 44 publications

The alteration of the C‐terminal region of human frataxin distorts its structural dynamics and function

The alteration of the C‐terminal region of human frataxin distorts its structural dynamics and function

Yeast Frataxin Sequentially Chaperones and Stores Iron by Coupling Protein Assembly with Iron Oxidation

Of the vulnerability of orphan complex proteins: The case study of the E. coli IscU and IscS proteins

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