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. 2010 Apr 13;8(4):e1000354.
doi: 10.1371/journal.pbio.1000354.

Structural basis for Fe-S cluster assembly and tRNA thiolation mediated by IscS protein-protein interactions

Rong Shi  1 , Ariane Proteau, Magda Villarroya, Ismaïl Moukadiri, Linhua Zhang, Jean-François Trempe, Allan Matte, M Eugenia Armengod, Miroslaw Cygler
Affiliations

Structural basis for Fe-S cluster assembly and tRNA thiolation mediated by IscS protein-protein interactions

Rong Shi et al. PLoS Biol. .

Abstract

The cysteine desulfurase IscS is a highly conserved master enzyme initiating sulfur transfer via persulfide to a range of acceptor proteins involved in Fe-S cluster assembly, tRNA modifications, and sulfur-containing cofactor biosynthesis. Several IscS-interacting partners including IscU, a scaffold for Fe-S cluster assembly; TusA, the first member of a sulfur relay leading to sulfur incorporation into the wobble uridine of several tRNAs; ThiI, involved in tRNA modification and thiamine biosynthesis; and rhodanese RhdA are sulfur acceptors. Other proteins, such as CyaY/frataxin and IscX, also bind to IscS, but their functional roles are not directly related to sulfur transfer. We have determined the crystal structures of IscS-IscU and IscS-TusA complexes providing the first insight into their different modes of binding and the mechanism of sulfur transfer. Exhaustive mutational analysis of the IscS surface allowed us to map the binding sites of various partner proteins and to determine the functional and biochemical role of selected IscS and TusA residues. IscS interacts with its partners through an extensive surface area centered on the active site Cys328. The structures indicate that the acceptor proteins approach Cys328 from different directions and suggest that the conformational plasticity of a long loop containing this cysteine is essential for the ability of IscS to transfer sulfur to multiple acceptor proteins. The sulfur acceptors can only bind to IscS one at a time, while frataxin and IscX can form a ternary complex with IscU and IscS. Our data support the role of frataxin as an iron donor for IscU to form the Fe-S clusters.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Network of protein-protein interactions involving IscS.
IscS initiates intracellular sulfur trafficking, delivering the sulfur to several sulfur-accepting proteins such as IscU, ThiI, TusA, and MoaD/MoeB that commit the sulfur to different metabolic pathways. IscU is the primary scaffold for assembly of Fe-S clusters. Frataxin/CyaY has been postulated as an Fe chaperone, an Fe donor for Fe-S cluster assembly, or a regulator of Fe-S cluster formation. In the schematic, sulfur delivering is indicated by red arrows and IscS-interacting proteins are framed by ovals (red, in sulfur accepting proteins).
Figure 2
Figure 2. Crystal structure of IscS complexes.
Cartoon representation of the IscS-TusA and IscS-IscU heterotetramers. The IscS subunits are colored cyan and green, TusA is magenta and IscU is orange. The Cys328 containing loops are red.
Figure 3
Figure 3. Interface between IscS and TusA or IscU.
(A) IscS-TusA, IscS (gray carbons), and TusA (yellow carbons). The Cys328IscS and Leu333IscS from the second subunit are shown with green carbons. The IscS residues in between are disordered. The conserved Asp45TusA and Asp51TusA are shown explicitly in stick mode. Hydrogen bonds are marked as dashed lines. Salt bridges Arg27TusA…Glu49IscS…Arg31TusA…Asp52IscS in the center of the interface and Glu21TusA…Arg220IscS at the periphery are explicitly shown; (B) IscS-IscU: IscU, gray carbons. The residues displayed are within 3.7 Å of its binding partner.
Figure 4
Figure 4. Small-angle X-ray scattering (SAXS) analysis of the complexes formed by IscS in solution.
Scattering data (desmeared, merged, and binned) are shown as squares and circles for the IscS-TusA and IscS-IscU complexes, respectively. The predicted scattering profiles calculated in CRYSOL from atomic coordinates are shown as plain black lines. The profiles were offset on the vertical axis for clarity.
Figure 5
Figure 5. Split open IscS-TusA interface (gray) with superposed uncomplexed proteins (yellow).
Above, TusA with secondary structure elements; below, IscS with semitransparent molecular surface. The reorientation of multiple sidechains creates better shape complementarity between the contacting molecular surfaces.
Figure 6
Figure 6. Protein binding surface of the IscS dimer.
(A) Residue conservation pattern on the surface of the IscS dimer. The view is toward the active site Cys328. The yellow line indicates the dimer interface and the yellow spheres mark the tips of the residues that have been mutated. The level of conservation of surface residues is marked in shades of burgundy (dark, high conservation; white, highly variable). The residues Cys328-Ser336 are colored cyan. (B) location of mutations affecting interaction with acceptor proteins: IscU, gray; TusA, blue; ThiI, in magenta; TusA/ThiI, cyan; CyaY/IscX/ThiI, dark green. The C-terminal residues 376–404 colored gray at the top-right are missing in the Δ376–404 deletion mutant. The footprint of IscU is marked by light blue line: TusA, yellow line; ThiI, red line; and IscX, CyaY, green line.
Figure 7
Figure 7. Interactions of IscS mutants with binding partners determined by in vitro pull-downs.
Only interactions of representative mutants are shown. The IscS is His-tagged, the partners are GST-tagged, and the mixture was loaded on the glutathione Sepharose, the beads washed, and analyzed by SDS-PAGE. The mutations are indicated above the lanes. Two lanes are shown for each mutant: left shown the mixture loaded on the column; right, proteins retained on the column. (A) TusA; (B) IscU. His-IscS and Gst-IscU appear at the same place on the SDS gel (left lane). To distinguish between them the proteins were released from the beads by TEV protease cleavage of the GST and elution of His-IscS and untagged IscU. Only A327V show a small decrease in the IscS/IscU ratio; (C) ThiI; (D) CyaY; (E) IscX.
Figure 8
Figure 8. The electrostatic potential of the IscS dimer: red, negative; blue, positive.
Surface with positive potential overlaps with the footprint of CyaY and IscX. Orientation similar to that in Figure 6B.
Figure 9
Figure 9. Modeling of the IscS-CyaY complex using the interface residues on CyaY identified by NMR and residues of IscS important for binding to CyaY as identified here.
The top 6 CyaY models are shown. The IscS subunits are painted green and slate. The overlapping CyaY models are shown in different colors. The locations of IscU (wheat) and TusA (red) relative to IscS are also shown. There are no steric conflicts between IscU and any of the CyaY models. The TusA molecule, however, clashes with all of the top models of CyaY, as was expected from the competition experiments.
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