Drosophila melanogaster Frataxin: Protein Crystal and Predicted Solution Structure with Identification of the Fe-Binding Regions
Andria V. Rodrigues, Sharon Batelu, Tiara V. Hinton, John Rotondo, Lindsey Thompson, Joseph S. Brunzelle, Timothy L. Stemmler [1]
Molecular Tour
Friedreich’s Ataxia (FRDA) is the most prevalent hereditary disease linked to failed iron-sulfur (Fe-S) cluster biosynthesis. FRDA is a human autosomal recessive genetic disorder caused by a trinucleotide expansion within the frataxin gene (FXN), which codes for the frataxin protein, FXN. The trinucleotide repeat results in an under expression of frataxin. This deficiency pathologically presents as mitochondrial iron overload, increased reactive oxygen species production, and a disruption in Fe-S cluster biosynthesis. Combined, these phenotypes are cytotoxic in metabolic tissues including the dorsal root ganglia and cardiomyocytes. FRDA presents in early adolescence as seen by positive ataxia, poor muscle coordination, and dysarthria. The protein frataxin is a key component in the mitochondrial iron-sulfur cluster bioassembly (ISC) pathway, where it serves as a modulator for cysteine desulfurase, and likely iron delivery to the scaffold. Cysteine desulfurase is the enzyme that provides sulfur, from L-Cysteine, to the scaffold protein for cluster assembly. In human cells, cysteine desulfurase (NFS1), coordinates with FXN and the accessory proteins, LYRM4 (ISD11) and acyl carrier protein (ACP1) to form a complex generating persulfide. The persulfide, as well as iron, is delivered to the scaffold protein (ISCU2) to complete [2Fe-2S] cluster assembly.
Recent reports from the Markley Lab (Cai, 2018) confirmed that Fe(II) is delivered by frataxin to the scaffold in the presence of ferredoxin; ferredoxin within the pathway provides reducing equivalents for cluster assembly. Previous reports (Kondapalli, 2008; Koebke, 2019) confirmed the binding affinities of Fe(II) to frataxin orthologs of yeast (Saccharomyces cerevisiae) and flies (Drosophila melanogaster). In Drosophila frataxin (Dfh), which has shown increased stability relative to its orthologs, iron binds within the micromolar affinity range. Other frataxin orthologs also bind in this range and these affinities are within the range of available Fe(II) in the mitochondria. This suggests that Dfh is loaded with Fe(II) while in the cellular mitochondrial matrix. Based on the iron binding capability of Dfh with respect to Fe-S cluster assembly and its increased stability, this report characterizes the crystal and solution state structures of Dfh and highlights likely Fe-binding residues.
In this study, we found that all frataxin orthologs are very similar in structure as expected from their comparative amino acid sequence alignment. However, this structural similarity does not translate to protein stability. , at a resolution of 1.4 Å, is a well-folded protein with a conserved αβ-sandwich motif with two α-helices, six β-sheets, and a 310-helix on the C-terminal tail. Labels for the different secondary structural elements are marked on the corresponding helix or strand (PDB ID: 7n9i). . This scene highlights the acidic patch on Dfh, and where iron is likely to bind to the protein. Remember to drag the structures with the mouse to rotate them. . The solution structure, which is highly similar, has minor differences in the length of the N-terminal tail and β-sheets, and the helix of the C-terminal tail is absent; differences likely due to crystal packing. Potential Fe-binding residues on Dfh were identified by nuclear magnetic resonance in the presence of Fe(II). Of the 133 residues in Dfh, 8 were perturbed, beyond the threshold, in the presence of iron. The residues are predominantly acidic and in the same region as seen in frataxin orthologs. Comparison of different orthologs of the frataxin protein:
These scenes emphasizes the conserved αβ-sandwich motif of frataxin across the orthologs.
Orientation of iron impacted residues on the crystal structure of Dfh:
References
- ↑ Rodrigues AV, Batelu S, Hinton TV, Rotondo J, Thompson L, Brunzelle JS, Stemmler TL. Drosophila melanogaster frataxin: protein crystal and predicted solution structure with identification of the iron-binding regions. Acta Crystallogr D Struct Biol. 2023 Jan 1;79(Pt 1):22-30. doi: , 10.1107/S2059798322011639. Epub 2023 Jan 1. PMID:36601804 doi:http://dx.doi.org/10.1107/S2059798322011639
