Three-Dimensional Structure and Determinants of Stability of the Iron-Sulfur Cluster Scaffold Protein IscU from Escherichia coli

Iron-sulfur (Fe-S) clusters are ancient and ubiquitous prosthetic groups involved in a wide variety of biological processes.1 In prokaryotes, the ISC (iron-sulfur cluster) biogenesis system, encoded by the isc operon (IscR, IscS, IscU, IscA, HscB, HscA, and ferredoxin) is mainly responsible for the ‘house-keeping’ generation of iron-sulfur clusters.2 The complex interactions of these proteins are essential for efficient Fe-S cluster biogenesis.3 A very similar ISC system is found in the mitochondria of eukaryotes.4 IscU is the scaffold protein for cluster assembly and transfer. The amino acid sequence of IscU is highly conserved over diverse organisms.2 More recently, it was found that a single intronic mutation in the human ISCU gene is associated with a myopathy resulting from the deficiencies in Fe-S proteins succinate dehydrogenase and aconitase.5,6 Complete removal of the iscu gene in mice resulted in an early embryonic death.7 Early NMR investigations of Thermotoga maritima IscU suggested that the apo-form of this protein has a ‘molten globule-like’ structure.8 IscU from Haemophilus influenzae showed a much less structured conformation in its apo-state than in its Zn2+-bound state.9 Thus, initial structural investigations of IscU focused on the Zn2+-bound form of the protein.9,10 The X-ray crystal structure of Aquifex aeolicus holo-IscU revealed a homotrimer with only one of the three subunits containing a [2Fe-2S] cluster. The two apo-IscU subunits adopted different conformations in the crystal.11 It has been reported that the IscU(D39A) substitution increases the stability of the Fe-S cluster in holo-IscU.12–14 We found that E. coli apo-IscU exists in solution as two interconverting conformations, one structured (S) and one dynamically disordered and lacking secondary structure (D).15,16 The two forms interconvert on time scale of about one second, and at pH 8.0 and 25 °C, the free energy between the two states is ~ 0.5 kcal/mol.16 A separate study from our group has demonstrated that the S⇆D transition involves the conversion of two peptidyl-prolyl peptide bonds from trans in the S-state to cis in the D-state (Dai Z, Tonelli, M, and Markley JL, submitted for publication). Thus, although the D-state appears disordered from the lack of dispersion of its backbone NMR chemical shifts,16 it does contain a fold that stabilizes two high-energy cis peptide bonds. The highly cooperative transition between the S- and D-states, which involves two concerted peptide bond isomerizations and a change in secondary structure, explain the high activation barrier and the consequent slow rates of interchange between the two states. Thus, IscU can be categorized as a metamorphic protein.17 Both states have found to be physiologically important. The D-state of IscU is the substrate for IscS, the PLP-dependent cysteine desulfurase that catalyzes the conversion of cysteine to alanine and transfers the sulfane sulfur generated to IscU.16 Once a cluster has been assembled, IscU converts to the S-state.11 The S-state of IscU binds preferentially to HscB (the DnaJ-like co-chaperone),15 and HscB targets the complex to HscA(ATP) (the DnaK-like chaperone in its ATP-bound state). Finally, upon ATP hydrolysis HscA(ADP) binds to the D-state of IscU ensuring cluster release to an acceptor protein (Kim JH et al., submitted for publication). IscU appears to have evolved to undergo the S⇆D interconversion and to have an optimal set point for this equilibrium. We found that certain single amino acid substitutions (D39A, N90A, S107A, E111A) stabilize the S-state of IscU, whereas others (K89A, N90D) stabilize the D-state.16 In vitro Fe-S cluster assembly studies showed that IscU variants with %S = (100×[S])/([S]+[D]) higher than wild-type (WT) assemble clusters slowly with an initial lag and that variants with %S lower than WT assemble less stable clusters at a slower rate without a lag.16 Thus it is of considerable interest to determine the structures of the S- and D-states along with the features of the IscU sequence that are responsible for its metamorphic properties and for setting the position of the equilibrium. We report here the solution structures of the S-states of apo-IscU(WT) and apo-IscU(D39A) determined by NMR spectroscopy. The structure of apo-IscU(WT) was determined under conditions in which %S ≈ 80; thus we had to contend with signals from the D-state of the protein. By contrast, apo-IscU(D39A) had %S ≈ 95. Comparison of structures revealed that the wild-type protein is more dynamic than the D39A variant, particularly in the cysteine-containing loops. This result suggests that the higher %S can be explained by stabilization of the S-state by the D39A substitution. The two structures have provided a framework for our analysis of data from several other single-site amino acid substitutions that alter %S.