Tangled web of interactions among proteins involved in iron-sulfur cluster assembly as unraveled by NMR, SAXS, chemical crosslinking, and functional studies

Abstract

Proteins containing iron-sulfur (Fe-S) clusters arose early in evolution and are essential to life. Organisms have evolved machinery consisting of specialized proteins that operate together to assemble Fe-S clusters efficiently so as to minimize cellular exposure to their toxic constituents: iron and sulfide ions. To date, the best studied system is the iron-sulfur cluster (isc) operon of Escherichia coli, and the eight ISC proteins it encodes. Our investigations over the past five years have identified two functional conformational states for the scaffold protein (IscU) and have shown that the other ISC proteins that interact with IscU prefer to bind one conformational state or the other. From analyses of the NMR spectroscopy-derived network of interactions of ISC proteins, small-angle X-ray scattering (SAXS) data, chemical crosslinking experiments, and functional assays, we have constructed working models for Fe-S cluster assembly and delivery. Future work is needed to validate and refine what has been learned about the E. coli system and to extend these findings to the homologous Fe-S cluster biosynthetic machinery of yeast and human mitochondria. This article is part of a Special Issue entitled: Fe/S proteins: Analysis, structure, function, biogenesis and diseases.

Keywords: Conformational equilibria; Iron–sulfur cluster biosynthesis; Nuclear magnetic resonance (NMR) spectroscopy; Protein–protein interactions; Small-angle X-ray scattering (SAXS).

Fig. 1

The isc operon and the ISC proteins it encodes.

Fig. 2

Network of protein–protein interactions involving IscU (purple) and IscS (green).

Fig. 3

Results from 2D ¹H–¹⁵N HSQC experiments that identified the nucleotide-dependent HscA binding surface upon HscB. [U–¹⁵N]–HscB was mixed with 3 molar equivalents of unlabeled HscA(T212V), which included a mutation that rendered the protein unable to cleave ATP, in the presence of either ATP (A) or ADP (B). Detailed NMR chemical shift perturbation data can be found in [90]. Depicted here is the structure of HscB (PDB 1fpo; [150]) with ATP- or ADP-HscA-induced ¹H^N–¹⁵N^H chemical shift perturbations (Δδ_NH) mapped onto the surface of HscB. In red are [U–¹⁵ N]-HscB ¹H^N–¹⁵N^H signals that could not be followed due to severe line broadening; in blue are signals with Δδ_NH > 0.01 ppm; and in black are signals with Δδ_NH < 0.01 ppm, or signals that were unassigned (e.g. proline residues). The insets in both (A) and (B) indicate a region of the J-domain that contains the highly conserved His-Pro-Asp (HPD) motif (HscB numbering: ³²HPD³⁴). Residues have been color coded as above, except that Pro³³ was colored gray to indicate its lack of NMR signal in the 2D ¹H–¹⁵N HSQC experiment. Copyright (2014) American Chemical Society.

Fig. 4

Schematic representation of conformational equilibrium of IscU. In its apo-state, IscU exists as a metamorphic protein, showing two different conformations; the more structured state (S-state) and the more dynamic state (D-state). This conformational transition is correlated with cis/trans isomerization of two peptidyl–prolyl peptide bonds, N13–P14 and P100–P101. Adapted from [39].

Fig. 5

Structural models of IscU in its S-state. (A) NMR solution structure of Haemophilus influenzae Zn-bound IscU (PDB 1r9p; [77]). Zn is shown as an orange sphere. Note that the disordered N-terminal region (M1–L20) is not shown here. (B) X-ray crystal structure of Aquifex aeolicus [2Fe–2S]-bound IscU (PDB 2z7e; [78]). The structure is trimeric with only one molecule in the asymmetric unit containing the [2Fe–2S] cluster (shown as spheres). (C) NMR solution structure of E. coli apo-IscU (PDB 2l4x; [102]). (Left) Representative structure. (Right) Ensemble of the 20 lowest-energy structural models.

Fig. 6

NMR evidence from 2D ¹H–¹⁵N HSQC spectra for the surfaces by which IscX and IscU interact. (A) Perturbation of the ¹H^N–¹⁵N^H signals (Δδ_NH) of [U–15N]–IscX, resulting from the addition of 4 equivalents of IscU. Red triangles denote residues whose chemical shift changes could not be followed because of severe line broadening. (B) Results from panel A mapped onto the structure of IscX (PDB 2bzt)¹⁷ with the following color code: gray, not affected; blue, significantly shifted (Δδ_NH > 0.04 ppm); red, broadened; black, not assigned or overlapped. (C) Perturbation of the ¹H^N–¹⁵N^H signals of [U–¹⁵N]–IscU resulting from the addition of 4 equivalents IscX. Red triangles denote residues whose chemical shift changes could not be followed because of severe line broadening. (D) Results from panel C mapped onto the structure of IscU (PDB 2l4x)³³ with the same color code used for panel B. Reprinted with permission from [70]. Copyright (2014) American Chemical Society.

Fig. 7

Structural models from SAXS data. (A) Experimental SAXS data (circles) recorded for IscS (blue), IscU + IscS (purple), IscX + IscS (red), and IscX + IscU + IscS (green). Fits of the molecular models for IscS (PDB 1p3w) and those determined from rigid body modeling to experimental SAXS are plotted as solid lines. (B) Pairwise distance distribution functions derived from experimental SAXS data (solid-lines) for IscS (blue), IscU + IscS (purple), IscX + IscS (red), and IscX + IscU + IscS (green) compared to those derived from the rigid body modeling derived structures (dashed-lines). (C) Molecular models of IscS (black, PDB 1p3w), IscU–IscS (purple), IscX–IscS (red), and IscX–IscU–IscS (green), determined from rigid body modeling simulations. The structures used for each complex component in the rigid body modeling simulations were: IscS (blue; PDB 1p3w) [137], IscU (purple; PDB 2l4x) [102], and IscX (red; PDB 2bzt) [56]. The resulting structures from rigid body modeling are overlaid with ab initio shape models determined from the SAXS data with DAMMIF [159]. Reprinted with permission from [70]. Copyright (2014) American Chemical Society.

Fig. 8

Proposed mechanism for the ISC Fe–S cluster assembly. In this scheme IscX serves both as the source of Fe²⁺ and as a regulator of cysteine desulfurase (IscS). The species ${\mathrm{S}}_{-}^{•}$ is bound to the sulfur of a cysteine residue of either IscS or IscU as indicated, and S²⁻:Fe³⁺ is bound to a cysteine residue of IscU as Cys–S–Fe–S. Details are provided in the text. Not shown is CyaY, which inhibits Fe–S cluster assembly by binding to IscS and competing off Fdx and IscU. Reprinted with permission from [70]. Copyright (2014) American Chemical Society.

Fig. 9

Schematic representation of the role of the S- and D-states of IscU in Fe–S cluster assembly on IscU and delivery to an acceptor apoprotein. (A) Reduced Fdx donates an electron to reduce the S⁰ generated by conversion of Cys to Ala by IscS. (B) The D-state of IscU binds to IscS displacing Fdx, and the sulfur radical is transferred from IscS to IscU and reduced by Fe²⁺ delivered by IscX to S²⁻. (C) Following the repeat of steps A and B, an assembled [2Fe–2S] cluster is bound to the S-state of IscU. (D) IscU:[2Fe–2S] is transferred from IscS to HscB, which has a higher affinity for the S-state of IscU than does IscS. (E) The J-domain of HscB (round knob) targets the HscB–IscU:[2Fe–2S] complex to the nucleotide binding domain of HscA. (F) The approach of an acceptor apoprotein (blue trapezoid) and subsequent attack of two of its –SH groups on the iron ions of the complex liberates two of the IscU cluster ligands, leading to a conformational change in HscB that activates the ATPase activity of HscA. (G) Cleavage of ATP to ADP leads to conversion of the tense state (T) state of HscA to the relaxed state (R), which binds the D-state of IscU. Conversion of IscU from the S- to the D-state by binding to HscA(ADP), releases the cluster to the acceptor protein, and HscB. (H) Finally, exchange of bound ADP for ATP converts HscA back to the T-state with release of IscU, which resumes its equilibrium between the S- and D-states. Adapted from [39] on the basis of new results [70]