ISCU(M108I) and ISCU(D39V) Differ from Wild-Type ISCU in Their Failure To Form Cysteine Desulfurase Complexes Containing Both Frataxin and Ferredoxin

Abstract

Whereas iron-sulfur (Fe-S) cluster assembly on the wild-type scaffold protein ISCU, as catalyzed by the human cysteine desulfurase complex (NIA)₂, exhibits a requirement for frataxin (FXN), in yeast, ISCU variant M108I has been shown to bypass the FXN requirement. Wild-type ISCU populates two interconverting conformational states: one structured and one dynamically disordered. We show here that variants ISCU(M108I) and ISCU(D39V) of human ISCU populate only the structured state. We have compared the properties of ISCU, ISCU(M108I), and ISCU(D39V), with and without FXN, in both the cysteine desulfurase step of Fe-S cluster assembly and the overall Fe-S cluster assembly reaction catalyzed by (NIA)₂. In the cysteine desulfurase step with dithiothreitol (DTT) as the reductant, FXN was found to stimulate cysteine desulfurase activity with both the wild-type and structured variants, although the effect was less prominent with ISCU(D39V) than with the wild-type or ISCU(M108I). In overall Fe-S cluster assembly, frataxin was found to stimulate cluster assembly with both the wild-type and structured variants when the reductant was DTT; however, with the physiological reductant, reduced ferredoxin 2 (rdFDX2), FXN stimulated the reaction with wild-type ISCU but not with either ISCU(M108I) or ISCU(D39V). Nuclear magnetic resonance titration experiments revealed that wild-type ISCU, FXN, and rdFDX2 all bind to (NIA)₂. However, when ISCU was replaced by the fully structured variant ISCU(M108I), the addition of rdFDX2 to the [NIA-ISCU(M108I)-FXN]₂ complex led to the release of FXN. Thus, the displacement of FXN by rdFDX2 explains the failure of FXN to stimulate Fe-S cluster assembly on ISCU(M108I).

Figure 1

(A) Comparison of the sequence-aligned C-terminal regions of eukaryotic and prokaryotic scaffold proteins indicating the conservation of a methionine residue in eukaryotes (red) and an isoleucine residue in prokaryotes (blue). Abbreviations: Ab, Acinetobacter baumannii; At, Arabidopsis thaliana; Av, Azotobacter vinelandii; Ba, Buchnera aphidicola; Dm, Drosophila melanogaster; Dr, Danio rerio; Ec, Escherichia coli; Hi, Haemophilus influenzae; Hs, Homo sapiens; Mm, Mus musculus; Nm, Neisseria meningitides; Rp, Rickettsia prowazekii; Sc, Saccharomyces cerevisiae; Tv, Trichomonas vaginalis. (B) NMR structures of the structured states of (green) mouse ISCU (PDB entry 1WFZ) and (yellow) H. influenzae IscU (PDB entry 1R9P) with the position of the differentially conserved residue highlighted.

Figure 2

Structural differences between ISCU and ISCU(M108I). (A) 2D ¹H–¹⁵N TROSY-HSQC spectrum of [U-¹⁵N]ISCU(M108I). (B) 2D ¹H–¹⁵N TROSY-HSQC spectrum of [U-¹⁵N]ISCU. (C) Overlay of spectra from panels A and B. (D) CD spectra of ISCU(M108I) (red) and ISCU (black).

Figure 3

¹H–¹⁵N TROSY-HSQC spectra annotated with assignments of backbone ¹H^N–¹⁵N^H signals of (A) ISCU(D39V) and (B) ISCU(M108I).

Figure 4

(A) Chemical shift differences between the ¹H–¹⁵N peaks of ISCU(D39V) and ISCU(M108I). (B) Chemical shift differences of the two ISCU variants mapped onto the structure of the NFS1–ISCU subcomplex (PDB entry 5WLW). Color code: gray, no significant differences; blue, large chemical shift differences (Δδ_NH > 1 ppm); black, no assignment.

Figure 5

Investigation of the interaction between ISCU(M108I) and (NIA)₂. 2D ¹H–¹⁵N TROSY-HSQC spectra of [U-¹⁵N]ISCU(M108I) after the addition of (A) 0.5 and (B) 1.0 subunit equivalent of (NIA)₂. (C) CS perturbation (Δδ_NH) of the ¹H–¹⁵N signals of [U-¹⁵N]ISCU(M108I) resulting from its interaction with (NIA)₂. The red triangles denote ¹H–¹⁵N peaks that are broadened beyond recognition. (D) CS perturbation from panel C mapped onto a portion of the structure of the NFS1–ISCU complex (PDB entry 5WLW). Color code: gray, not significantly affected (Δδ_NH < 0.1 ppm); blue, significant chemical shift changes (Δδ_NH > 0.1 ppm); red, severe line broadening; black, no assignments. (E) Size-exclusion chromatography (SEC) profiles of (NIAU(M108I))₂ (red solid trace), (NIAU)₂ (black dashed trace), and (NIA)₂ (blue dashed trace). (F) SDS–PAGE analysis the SEC fraction (indicated by a red arrow pointing to the red trace in panel E) of (NIAU(M108I))₂ (note that the Acp band is weak because it stains weakly). (G) ITC analysis of the interactions between (NIA)₂ and ISCU(M108I). The top panels show peaks indicating heat released after each injection; the bottom panels show data points fitted to a single 1:1 binding constant to yield thermodynamic parameters.

Figure 6

Evidence that the FXN bypassing phenotype of ISCU depends on the reducing agent. (A) Cysteine desulfurase activity assay of (NIA)₂. (B) Fe–S cluster reconstitution catalyzed by (NIA)₂ on ISCU, ISCU(M108I), and ISCU(D39V), with or without FXN, using DTT as the reducing agent. (C) Fe–S cluster reconstitution catalyzed by (NIA)₂ on ISCU, ISCU(M108I), and ISCU(D39V), with or without FXN, using rdFDX2 as the reducing agent. The conditions of each experiment are indicated in the figure.

Figure 7

Quantification of Fe–S cluster assembly rates on ISCU variants with (A) DTT or (B) rdFDX2 as the reducing agent.

Figure 8

NMR spectra showing that FDX2 binds to (NIAU)₂–FXN₂ without displacement of FXN but that FDX2 added to (NIAU(M108I))₂–FXN₂ displaces FXN. (A) ¹H–¹⁵N TROSY-HSQC spectrum of [U-¹⁵N]FXN. (B) ¹H–¹⁵N TROSY-HSQC spectrum of [U-¹⁵N]FXN after the addition of 0.5 subunit equivalent of unlabeled (NIAU)₂. (C) ¹H–¹⁵N TROSY-HSQC spectrum of [U-¹⁵N]FXN after the addition of 0.5 subunit equivalent of unlabeled (NIAU)₂ and 1.0 subunit equivalent of unlabeled FDX2. (D) ¹H–¹⁵N TROSY-HSQC spectrum of [U-¹⁵N]FXN. (E) ¹H–¹⁵N TROSY-HSQC spectrum of [U-¹⁵N]FXN after the addition of 0.5 subunit equivalent of unlabeled (NIAU(M108I))₂. (F) ¹H–¹⁵N TROSY-HSQC spectrum of [U-¹⁵N]FXN after the addition of 0.5 subunit equivalent of unlabeled (NIAU(M108I))₂ followed by the addition of 1.0 subunit equivalent of unlabeled FDX2.