Figure 4.

(a) (Top) ¹H–¹⁵N HSQC spectra of IscU (left) showing duplicated peaks for the lone Trp76 sidechain (dotted box) that indicate the co-existence of two conformations in the sample. Overlays of spectra (middle) of IscU in the presence of 3 mM Zn²⁺ (red) or D39A IscU (green) with apo wt IscU (black) show that the minor Trp sidechain resonance disappears in both cases. This indicates that Zn²⁺ and the D39A mutation stabilize the same conformation (IscU-S). (Bottom) ¹⁵N magnetization exchange spectrum of apo wt IscU that shows cross-peaks (red) between the diagonal sidechain resonances originating from Trp76 in the two co-existing conformations of IscU. The appearance of these cross-peaks demonstrates that IscU-S and IscU-D interconvert reversibly on the ms–s timescale. (b) Cartoon representation of the NMR structure of IscU-S (PDB ID: 2L4X). The four Pro residues in IscU-S are shown as red sticks, while the aromatic residues are coloured magenta. Curiously, most of the aromatic residues are solvent-exposed and do not contribute to the hydrophobic core of IscU-S. The structured IscU-S is in equilibrium on the ms–s timescale with a partially disordered form, IscU-D. Two of the Xaa-Pro linkages in IscU-D, Asn13–Pro14 and Pro100–Pro101 are in cis conformation, while all four Xaa-Pro bonds in IscU-S are in the trans form. (c) The role of the scaffold protein IscU in the functional Fe–S cluster biogenesis cycle. (1) IscU exists in two conformations, IscU-S and IscU-D. (2) The pyridoxal phosphate (PLP)-dependent enzyme IscS catalyzes the conversion of Cys to Ala and in-turn generates persulfides on IscU-D by preferentially binding to it. Once Fe is introduced into this complex (3), fold-switching occurs and the IscU-state is stabilized by the Fe–S cluster. The J-domain co-chaperone, HscB, recognizes IscU-S and facilitates the assembly and transfer of the Fe–S cluster to the downstream acceptor protein (5–9). During this process, IscU is transferred to the Hsp70-like HscA, which stabilizes the partially disordered IscU-D form of IscU. Upon release from HscA, free IscU then equilibrates again into ordered and partially unfolded forms to reset the cycle. Panels (a) and (c) are reproduced with permission from Markley et al. [17].