Yeast frataxin solution structure, iron binding, and ferrochelatase interaction

Abstract

The mitochondrial protein frataxin is essential for cellular regulation of iron homeostasis. Although the exact function of frataxin is not yet clear, recent reports indicate the protein binds iron and can act as a mitochondrial iron chaperone to transport Fe(II) to ferrochelatase and ISU proteins within the heme and iron-sulfur cluster biosynthetic pathways, respectively. We have determined the solution structure of apo yeast frataxin to provide a structural basis of how frataxin binds and donates iron to the ferrochelatase. While the protein's alpha-beta-sandwich structural motif is similar to that observed for human and bacterial frataxins, the yeast structure presented in this report includes the full N-terminus observed for the mature processed protein found within the mitochondrion. In addition, NMR spectroscopy was used to identify frataxin amino acids that are perturbed by the presence of iron. Conserved acidic residues in the helix 1-strand 1 protein region undergo amide chemical shift changes in the presence of Fe(II), indicating a possible iron-binding site on frataxin. NMR spectroscopy was further used to identify the intermolecular binding interface between ferrochelatase and frataxin. Ferrochelatase appears to bind to frataxin's helical plane in a manner that includes its iron-binding interface.

Figure 1

¹H–¹⁵N HSQC spectrum of ¹⁵N-labeled frataxin. The spectrum was collected at 30 °C on a Varian INOVA 600 MHz NMR spectrometer. Corresponding peak assignments are given adjacent to spectral features. Side chain amine signals are designated with asterisks and connected to partners by a line when applicable. Aliased peaks are designated with pound signs.

Figure 2

Ribbon drawing (A) and electrostatic surface plots (B) for frataxin at three different orientations around the vertical axis. (A) Labels for the different secondary structural elements are given adjacent to the corresponding helix or strand. (B) At the left is a surface electrostatic potential plot for frataxin at a molecular orientation identical to that shown in panel A. The surface was calculated using a probe radius of 1.4 Å and the potential surface displayed with a scale from −10 to 10 kbT using MOLMOL. At the right, the top and bottom panels correspond to a rotation of the central surface about the vertical axis by −120° and 120°, respectively. To orient the reader, secondary structural element landmarks have been labeled.

Figure 3

NMR chemical shift mapping of frataxin residues that interact with iron. (A) ¹H–¹⁵N HSQC spectra for frataxin in the presence (red) and absence (black) of iron. The iron spectrum was collected at a 2:1 iron:protein stoichiometric ratio. Peaks boxed with dashed lines are present only in the iron spectrum at low threshold levels. The region boxed with a solid line is expanded in Figure 4B. (B) Expansion of the region in the ¹⁵N HSQC spectrum for frataxin in the presence (red) and absence (black) of iron. (C) Residues identified on the apofrataxin structure that have normalized chemical shift (δ) values greater than 1 (colored red).

Figure 4

Iron-induced amide chemical shift perturbations at varying iron:frataxin concentration ratios. Frataxin secondary structure information is given at the top. Perturbations in backbone ¹H and ¹⁵N resonances, induced by the addition of Fe(NH₄)₂(SO₄)₂ to ¹⁵N-labeled apofrataxin, at the following iron:protein stoichiometries are plotted: (A) 2:1, (B) 1.5:1, (C) 1:1, and (D) 0.5:1.

Figure 5

NMR chemical shift mapping of the ferrochelatase binding surface on frataxin. (A) Chemical shift-adjusted TROSY ¹H–¹⁵N HSQC spectra for frataxin in the presence (red) and absence (black) of ferrochelatase at a 1:1 frataxin:ferrochelatase stoichiometry. (B) Expansion of the region identified in the spectra to the left. (C) Residues identified on the apofrataxin structure that have normalized chemical shift (δ) values greater than 1 (colored red).

Figure 6

Identification of residues at yeast frataxin's ferrochelatase binding interface. Frataxin secondary structure information is given at the top. Perturbations in backbone ¹H and ¹⁵N resonances were induced by the addition of ferrochelatase to ¹⁵N-labeled apofrataxin at ferrochelatase:protein concentration ratios of 1:1. Normalized chemical shift changes are plotted vs amino acid sequence number.

Figure 7

Alignment of structurally characterized frataxin orthologs. Multiple-sequence alignment of frataxin orthologs of known structure, displayed in ClustalX colors, corresponding to all residues in mature yeast frataxin (43, 44) as well as the structurally characterized regions of full-length bacterial and truncated mature human frataxin. Vertical lines in the human N-terminal sequence represent residues omitted from the mature human frataxin structures. The numbering refers to yeast frataxin. Secondary structural elements corresponding to frataxin are given directly above the sequences.