[2Fe-2S]-Ferredoxin Binds Directly to Cysteine Desulfurase and Supplies an Electron for Iron–Sulfur Cluster Assembly but Is Displaced by the Scaffold Protein or Bacterial Frataxin

Escherichia coli [2Fe-2S]-ferredoxin (Fdx) is encoded by the <i>isc</i> operon along with other proteins involved in the ‘house-keeping’ mechanism of iron–sulfur cluster biogenesis. Although it has been proposed that Fdx supplies electrons to reduce sulfane sulfur (S⁰) produced by the cysteine desulfurase (IscS) to sulfide (S^2–) as required for the assembly of Fe–S clusters on the scaffold protein (IscU), direct experimental evidence for the role of Fdx has been lacking. Here, we show that Fdx (in either oxidation state) interacts directly with IscS. The interaction face on Fdx was found to include residues close to its Fe–S cluster. In addition, C328 of IscS, the residue known to pick up sulfur from the active site of IscS and deliver it to the Cys residues of IscU, formed a disulfide bridge with Fdx in the presence of an oxidizing agent. Electrons from reduced Fdx were transferred to IscS only in the presence of l-cysteine, but not to the C328S variant. We found that Fdx, IscU, and CyaY (the bacterial frataxin) compete for overlapping binding sites on IscS. This mutual exclusion explains the mechanism by which CyaY inhibits Fe–S cluster biogenesis. These results (1) show that reduced Fdx supplies one electron to the IscS complex as S⁰ is produced by the enzymatic conversion of Cys to Ala and (2) explain the role of Fdx as a member of the <i>isc</i> operon

Statistics for the NMR Structure of C2 2A^pro.

a<p>Stretches of regular secondary structure: 7–9, 12–16, 28–30, 35–39, 55–60, 65–74, 78–79, 88–96, 108–110, 115–122, 127–131.</p

DNA Primers used for Cloning and Mutating RV-C2 2A^pro.

<p>*Restriction sites are in bold; primer regions that anneal to 2A^pro gene are underlined; and lowercase letters show DNA bases at the sites of directed mutagenesis.</p

Cross-eyed stereoscopic representations of 2A^pro structures.

<p>(A) Superimposition of backbones of the four proteases showing their structural similarity. Pairwise rmsd values for C2 relative to both A2 and CB4 proteases are both 1.809 Å, while to EV71 protease is 1.4 Å. Poisson-Boltzmann electrostatic potential surfaces are illustrated by PyMOL <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0097198#pone.0097198-Shin1" target="_blank">[29]</a> for (B) C2, (C) A2,(D) CB4 and (E) EV71 2A^pro. Each structure is shown in the same orientation. (F) Comparison of the positions of the bll−cll and cll−dll loops in the structures of C2 (<i>blue</i>), A2 (<i>red</i>), CB4 (<i>green</i>), and EV71 (<i>orange</i>) 2A^pro.</p

An RV RNA genome encodes a single polyprotein.

<p>The polyprotein is cleaved co- and post-translationally to release mature viral proteins. During infection, 2A^pro is excised at the N-terminus by self-catalysis and at the C-terminus by 3C^pro. The released protease cleaves cellular substrates including eIF4G and nucleoporins.</p

Properties of C2 2A^pro datasets.

<p>(A) Secondary structural features from the NMR solution structure: β-strands (<i>arrows</i>) and 3₁₀ helices (<i>boxes</i>). (B) The total number of constraints used for the structure calculation plotted as a function of residue number. (C) Rmsd values for backbone atoms (N, C^α, and C′) of the best 15 models relative to the average structure. Structurally compact regions have rmsd values below 2 Å.</p

Solution structure of C2 2A^pro.

<p>(A) The backbone atoms (N, C^α, C′) for the best 15 models as superimposed by MOLMOL<i>³¹</i> for the regions of regular secondary structure. (B) Ribbon diagram of the lowest energy model indicating the N-terminal domain (<i>orange</i>), C-terminal domain (<i>gray</i>), and the connecting loop (<i>green</i>). Stick representations (<i>magenta</i>) show the side chains (C₅₁, C₅₃, C₁₁₁, H₁₁₃) ligating the zinc ion (<i>gray sphere</i>), and side chains of the residues (<i>cyan</i>) forming the catalytic triad (H₁₈, D₃₄, C₁₀₅A). The di-tyrosine flap (Y₈₄, Y₈₅, P₈₆) lies near this triad. The two structures are rotated by 180^o.</p

¹H-¹⁵N HSQC spectra of ¹⁵N-labeled wild-type 2A^pro (<i>purple</i>) and C₁₀₅A 2A^pro (<i>red</i>).

<p>The two spectra are very similar; however, that of the wild-type protease exhibits small signals attributed to self-cleavage products.</p

SDS-PAGE illustrating purification of RV-C2 2A^pro.

<p>The recombinant methods described above were used to prepare ¹³C/¹⁵N-labeled C2 2A^pro (C₁₀₅A) for NMR studies. Representative samples from the procedure were fractionated by SDS-PAGE then visualized with Bio-Rad Stain-Free. Lane 1, Bio-Rad Precision Plus protein standards; lane 2, protein pellet after (NH₄)₂SO₄ precipitation; lane 3, SUMO-2A^pro after IMAC elution; lane 4, 2Apro after SUMO cleavage and IMAC elution; lanes 5–6, final protein fractions after gel filtration.</p