We have identified several single-site amino acid substitutions that shift the equilibrium between the S and D states of apo-IscU. Some substitutions favor the S state and some the D state. (A) Amino acid sequence of E. coli IscU: (black highlight) absolute conservation, (blue) conservative substitutions, and (yellow) semiconservative substitutions. Red arrows indicate positions substituted in the variants reported here. (B and C) Two-dimensional ¹H-¹⁵N NMR spectra of E. coli IscU variant proteins labeled uniformly with nitrogen-15. The spectra in B show that IscU variants D39A, N90A, S107A, and E111A are largely structured, whereas those in C show that IscU variants K89A and N90D are largely disordered. (D) Thermal stabilities of wild-type apo-IscU (WT) and variants (D39A, S107, E111A, and N90A) that favor the S state. The variants that favor the D state, being largely unfolded, did not exhibit a thermal transition.

IscS binds preferentially to and stabilizes the D state of apo-IscU. (A) Volume of the S → D (Left and Center for Asn26 and Glu95, respectively) or D → S (Right for Asn26) exchange cross peak divided by the volume of the diagonal S or D peak, respectively, as a function of the mixing time in a two-dimensional NMR exchange experiment. Blue symbols are for IscU alone; green symbols are for IscU plus 0.1 equivalent of IscS. From the initial slopes, the S → D exchange rate increased from 0.77 s^-1 for IscU alone to 1.1 s^-1 for IscU in the presence of IscS; the D → S exchange rate decreased from 2.0 s^-1 for IscU alone to 1.5 s^-1 for IscU in the presence of IscS. The D⇆S equilibrium constant for apo-IscU from these measurements (0.24) is consistent with the relative populations of the S and D species under these conditions. (B) Two-dimensional ¹H-¹⁵N NMR spectrum of apo-IscU labeled uniformly with nitrogen-15 (Left; red) and the spectrum of the same sample mixed with a stoichiometric amount of unlabeled IscS (Right; blue). (C) Extensive NMR assignments of signals from apo-IscU in the apo-IscU:IscS complex show that these IscU residues are disordered in the complex. The graphs show absolute values of the difference between the normalized backbone amide ¹H-¹⁵N chemical shifts in the apo-IscU:IscS complex and those in the D (Upper) and S (Lower) state of free apo-IscU. For comparison, the data from the Upper panel (open triangle) are plotted on the same scale in the Lower panel. Red X’s in the Upper panel indicate residues whose signals were observed as apo-IscU(D) but broadened and disappeared upon addition of IscS. The normalized chemical shifts were calculated by the formula Δδ_NH = [(Δδ_N/6)² + (Δδ_H)²]^1/2. The chemical shifts of the observed residues in the complex were similar to those of the D state and very different from those of the S state of apo-IscU. (D) X-ray crystal structure (Protein Data Bank ID code 3LVL) of the IscU:IscS complex (two views at right angles on one another) with IscU color coded to report information from NMR spectroscopy: Green are IscU residues determined to be disordered in the complex in solution on the basis of assigned NMR chemical shifts; red are residues observed in free apo-IscU that broadened and disappeared upon addition of IscS; and black are those residues whose NMR signals were not observed in either free apo-IscU or in the complex. Residues in yellow are from IscS.

Time course of iron-sulfur cluster assembly as monitored by absorbance at 456 nm. WT apo-IscU assembled clusters more efficiently than the other five variants studied. The more structured variants (E111A, S107A, and N90A) assembled clusters following an initial lag and at a slower rate than WT. Like WT, the less structured variants (K89A and N90D) did not exhibit an initial lag, but they assembled clusters at rates intermediate between WT and the more structured variants. The decay of intensity after its highest point arises from the instability of the cluster once formed.

Working model for iron-sulfur cluster assembly. (A) The scaffold protein, IscU, is monomeric and populates two conformational states in solution, one S and one D, that yield separate sets of NMR signals. In both states, the N-terminal residues (represented in red) are disordered, and a single set of NMR signals is observed. (B) The cysteine desulfurase, IscS, is a homodimer. (C) Upon binding IscS, IscU becomes largely disordered. (D) The cysteine desulfurase converts l-cysteine to l-alanine and in doing so generates sulfur, which is picked up by mobile cysteine residues of IscU and, along with iron ions, is used in cluster assembly.