Cellular depletion of the human protein frataxin is correlated with the neurodegenerative disease Friedreich's ataxia and results in the inactivation of Fe-S cluster proteins. Most researchers agree that frataxin functions in the biogenesis of Fe-S clusters, but its precise role in this process is unclear. Here we provide in vitro evidence that human frataxin binds to a Nfs1, Isd11, and Isu2 complex to generate the four-component core machinery for Fe-S cluster biosynthesis. Frataxin binding dramatically changes the K(M) for cysteine from 0.59 to 0.011 mM and the catalytic efficiency (k(cat)/K(M)) of the cysteine desulfurase from 25 to 7900 M⁻¹s⁻¹. Oxidizing conditions diminish the levels of both complex formation and frataxin-based activation, whereas ferrous iron further stimulates cysteine desulfurase activity. Together, these results indicate human frataxin functions with Fe(2+) as an allosteric activator that triggers sulfur delivery and Fe-S cluster assembly. We propose a model in which cellular frataxin levels regulate human Fe-S cluster biosynthesis that has implications for mitochondrial dysfunction, oxidative stress response, and both neurodegenerative and cardiovascular disease.
Fe-S clusters are partners in the origin of life that predate cells, acetyl-CoA metabolism, DNA, and the RNA world. The double helix solved the mystery of DNA replication by base pairing for accurate copying. Yet, for genome stability necessary to life, the double helix has equally important implications for damage repair. Here we examine striking advances that uncover Fe-S cluster roles both in copying the genetic sequence by DNA polymerases and in crucial repair processes for genome maintenance, as mutational defects cause cancer and degenerative disease. Moreover, we examine an exciting, controversial role for Fe-S clusters in a third element required for life – the long-range coordination and regulation of replication and repair events. By their ability to delocalize electrons over both Fe and S centers, Fe-S clusters have unbeatable features for protein conformational control and charge transfer via double-stranded DNA that may fundamentally transform our understanding of life, replication, and repair.
Iron–sulfur clusters are ubiquitous protein cofactors with critical cellular functions. The mitochondrial Fe–S assembly complex, which consists of the cysteine desulfurase NFS1 and its accessory protein (ISD11), the Fe–S assembly protein (ISCU2), and frataxin (FXN), converts substrates l-cysteine, ferrous iron, and electrons into Fe–S clusters. The physiological function of FXN has received a tremendous amount of attention since the discovery that its loss is directly linked to the neurodegenerative disease Friedreich’s ataxia. Previous in vitro results revealed a role for human FXN in activating the cysteine desulfurase and Fe–S cluster biosynthesis activities of the Fe–S assembly complex. Here we present radiolabeling experiments that indicate FXN accelerates the accumulation of sulfur on ISCU2 and that the resulting persulfide species is viable in the subsequent synthesis of Fe–S clusters. Additional mutagenesis, enzyme kinetic, UV–visible, and circular dichroism spectroscopic studies suggest conserved ISCU2 residue C104 is critical for FXN activation, whereas C35, C61, and C104 are all essential for Fe–S cluster formation on the assembly complex. These results cannot be fully explained by the hypothesis that FXN functions as an iron donor for Fe–S cluster biosynthesis, and further support an allosteric regulator role for FXN. Together, these results lead to an activation model in which FXN accelerates persulfide formation on NFS1 and favors a helix-to-coil interconversion on ISCU2 that facilitates the transfer of sulfur from NFS1 to ISCU2 as an initial step in Fe–S cluster biosynthesis.
Point estimates calculated from eligible studies showed that the three mPCRs (FilmArray, Verigene RV+ and ProFlu+) are highly accurate and may provide important diagnostic information for early identification of respiratory virus infections. In patients with low pretest probability for FluA, these three mPCRs can predict a low possibility of infection and may justify withholding empirical antiviral treatments.
The role of dolichol phosphate in the synthesis of N-glycosidically linked oligosaccharides of glycoproteins is well documented (see Refs. 1 and 2 for recent reviews). These glycoproteins possess a common core region ( 2 N-acetylglucosamine and at least 3 mannose residues) synthesized via the dolichol phosphate pathway. An oligosaccharide is preformed on dolichol phosphate, transferred to an asparagine residue in the protein, and then extensively modified to yield a heterogeneous population of N-linked oligosaccharides.Another class of glycoconjugates, composed of most of the proteoglycans, also contains a core oligosaccharide structure. This core is a tetrasaccharide (glucuronic acid-galactose-gaGrants 5R01 NS15775-02 and AM-05816. This paper is part of the
Friedreich’s ataxia (FRDA) is a progressive neurodegenerative disease that has been linked to defects in the protein frataxin (Fxn). Most FRDA patients have a GAA expansion in the first intron of their Fxn gene that decreases protein expression. Some FRDA patients have a GAA expansion on one allele and a missense mutation on the other allele. Few functional details are known for the ~15 different missense mutations identified in FRDA patients. Here in vitro evidence is presented that indicates the FRDA I154F and W155R variants bind more weakly to the complex of Nfs1, Isd11, and Isu2 and thereby are defective in forming the four-component SDUF complex that constitutes the core of the Fe-S cluster assembly machine. The binding affinities follow the trend Fxn ~ I154F > W155F > W155A ~ W155R. The Fxn variants also have diminished ability to function as part of the SDUF complex to stimulate the cysteine desulfurase reaction and facilitate Fe-S cluster assembly. Four crystal structures, including the first for a FRDA variant, reveal specific rearrangements associated with the loss of function and lead to a model for Fxn-based activation of the Fe-S cluster assembly complex. Importantly, the weaker binding and lower activity for FRDA variants correlate with the severity of disease progression. Together, these results suggest Fxn triggers sulfur transfer from Nfs1 to Isu2, and that these in vitro assays are sensitive and appropriate for deciphering functional defects and mechanistic details for human Fe-S cluster biosynthesis.
SummaryArchaea employ the archaellum, a type IV pilus-like nanomachine, for swimming motility. In the crenarchaeon Sulfolobus acidocaldarius, the archaellum consists of seven proteins: FlaB/X/G/F/H/I/J. FlaF is conserved and essential for archaellum assembly but no FlaF structures exist. Here, we truncated the FlaF N terminus and solved 1.5-Å and 1.65-Å resolution crystal structures of this monotopic membrane protein. Structures revealed an N-terminal α-helix and an eight-strand β-sandwich, immunoglobulin-like fold with striking similarity to S-layer proteins. Crystal structures, X-ray scattering, and mutational analyses suggest dimer assembly is needed for in vivo function. The sole cell envelope component of S. acidocaldarius is a paracrystalline S-layer, and FlaF specifically bound to S-layer protein, suggesting that its interaction domain is located in the pseudoperiplasm with its N-terminal helix in the membrane. From these data, FlaF may act as the previously unknown archaellum stator protein that anchors the rotating archaellum to the archaeal cell envelope.
DNA metabolism and processing frequently require transient or metastable DNA conformations that are biologically important but challenging to characterize. We use gold nanocrystal labels combined with small angle X-ray scattering to develop, test, and apply a method to follow DNA conformations acting in the Escherichia coli mismatch repair (MMR) system in solution. We developed a neutral PEG linker that allowed gold-labeled DNAs to be flashcooled and stored without degradation in sample quality. The 1,000-fold increased gold nanocrystal scattering vs. DNA enabled investigations at much lower concentrations than otherwise possible to avoid concentration-dependent tetramerization of the MMR initiation enzyme MutS. We analyzed the correlation scattering functions for the nanocrystals to provide higher resolution interparticle distributions not convoluted by the intraparticle distribution. We determined that mispair-containing DNAs were bent more by MutS than complementary sequence DNA (csDNA), did not promote tetramer formation, and allowed MutS conversion to a sliding clamp conformation that eliminated the DNA bends. Addition of second protein responder MutL did not stabilize the MutS-bent forms of DNA. Thus, DNA distortion is only involved at the earliest mispair recognition steps of MMR: MutL does not trap bent DNA conformations, suggesting migrating MutL or MutS/MutL complexes as a conserved feature of MMR. The results promote a mechanism of mismatch DNA bending followed by straightening in initial MutS and MutL responses in MMR. We demonstrate that small angle X-ray scattering with gold labels is an enabling method to examine protein-induced DNA distortions key to the DNA repair, replication, transcription, and packaging. D NA is frequently considered a passive component in interactions with proteins involved in DNA metabolism. Despite this view, many proteins use DNA structural features to mediate catalysis and identify damaged DNA through the effects of damage on DNA rigidity and conformation (1-6). The view of DNA as a passive element is therefore at least in part due to a paucity of robust tools to examine dynamic DNA conformational states during multistep reactions. Gold-labeled DNA enables measurement over length scales sufficient to accommodate several proteins to identify cooperative effects on DNA. Because X-rays scatter predominantly from electrons, using heavy atom labels (7-11) provides high contrast relative to organic molecules. By using labels of moderate size (∼5 nm), the scattering from gold nanocrystals dominates all other scattering signals by three orders of magnitude, thereby reducing analysis complexity while minimizing nanocrystal influence on biological macromolecules. Importantly, small angle X-ray scattering (SAXS) provides global information on conformations adopted by a population of macromolecules in almost any solution condition (12)(13)(14).Mismatch repair (MMR) is an evolutionarily conserved process that corrects mismatches generated during DNA replication (15,16). Despite the i...
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