Identification of IscU residues critical for de novo iron–sulfur cluster assembly

Abstract

Summary IscU is a central component of the ISC machinery and serves as a scaffold for the de novo assembly of iron–sulfur (Fe–S) clusters prior to their delivery to target apo‐Fe–S proteins. However, the molecular mechanism is not yet fully understood. In this study, we have conducted mutational analysis of E. coli IscU using the recently developed genetic complementation system of a mutant that can survive without Fe–S clusters. The Fe–S cluster ligands (C37, C63, H105, C106) and the proximal D39 and K103 residues are essential for in vivo function of IscU and could not be substituted with any other amino acids. Furthermore, we found that substitution of Y3, a strictly conserved residue among IscU homologs, abolished in vivo functions. Surprisingly, a second‐site suppressor mutation in IscS (A349V) reverted the defect caused by IscU Y3 substitutions. Biochemical analysis revealed that IscU Y3 was crucial for functional interaction with IscS and sulfur transfer between the two proteins. Our findings suggest that the critical role of IscU Y3 is linked to the conformational dynamics of the flexible loop of IscS, which is required for the ingenious sulfur transfer to IscU.

Evidence for dynamic in vivo interconversion of the conformational states of IscU during iron–sulfur cluster biosynthesis

“…These substitutions were scattered over the whole molecule of IscU; internal/surface molecule, loop/secondary structure, and proximal/distal to the active site for Fe-S cluster assembly, suggesting that the bypassing activity cannot be ascribed to interaction with a specific protein. We note that the essential residues of IscU (Tyr3, Cys37, Asp39, Cys63, Lys103, His105, and Cys106) (Johnson et al, 2006;Tanaka et al, 2019) remained unchanged, making it unlikely that interaction with IscS and assembly of Fe-S clusters was compromised by these substitutions. This view is supported by the observation that, in the presence of HscBA, cells carrying the F20C, N26I, V27F, G30V, or R57S suppressor mutation exhibited normal growth (Figure 2b), indicating that the scaffold function was not abrogated in these altered proteins.…”

“…Importantly, the structure of the D-shifted variants (Group 1: F20C, G28V, T74N, and Group 2: N26I, G30V, L97I, L99Q) could be restored to the S-state in the presence of Zn 2+ (Figure 4). This is probably because the ligand residues of the Fe-S cluster (Cys37, Asp39, Cys63, His105, and Cys106) (Shimomura et al, 2008;Tanaka et al, 2019), which had been spatially dispersed in the D-state, moved close to each other by binding to Zn 2+ , thereby stabilizing the S-state (Iannuzzi et al, 2014). By contrast, this conformational restoration was not observed for V27F (Group 2) and A85E (Group 1) variants, whose CD spectra were the same in the presence and absence of ZnSO 4 .…”

“…Group 1a IscU is distributed in alpha-, beta-, and gamma-proteobacteria, and also eukaryotic mitochondria, together with the components of the canonical ISC machinery (IscS, IscA, HscB, HscA, and Fdx). Amino acid residues critical for Group 1a IscU (Tyr3, Cys37, Asp39, Cys63, Lys103, His105, and Cys106) (Johnson et al, 2006;Tanaka et al, 2019) are also conserved in almost all sequences in the Group 1c IscU-like subfamily (as well as the Group 1b NifU subfamily), suggesting that they are also involved in de novo Fe-S assembly (Figure S5). With the exception of IscS, however, auxiliary components of the canonical ISC machinery (IscA, HscB, and Fdx; the situation with HscA is unclear because of difficulties distinguishing it from the ubiquitous DnaK chaperone) are rarely encoded in the genomes harboring the Group 1c subfamily (Yokoyama et al, 2018).…”

Molecular Details of the Frataxin–Scaffold Interaction during Mitochondrial Fe–S Cluster Assembly

“…Several residues have been shown to support the structural and biochemical integrity of the scaffold molecule. The N-terminal tyrosine (human Y 35 ) is important for internal stability of the bacterial scaffold (Figure 6D) [77]. This residue is important for stabilizing the interaction between the scaffold and cysteine desulfurase; the bacterial Y 35 -equivalent interacts with bacterial cysteine desulfurase through a hydrogen bond from the Tyr aromatic hydroxyl unit.…”

“…This residue is important for stabilizing the interaction between the scaffold and cysteine desulfurase; the bacterial Y 35 -equivalent interacts with bacterial cysteine desulfurase through a hydrogen bond from the Tyr aromatic hydroxyl unit. In bacteria, when this residue was substituted with 19 different amino acids, only the Y 35 F, H and W mutations were functionally equivalent, albeit cells grew at lower growth levels, indicating that aromatic or imidazole ring atoms at this ISCU position are essential for protein stability [77]. In addition to the ISCU's N-terminal tyrosine, humanequivalent residues C 69 , D 71 and H 137 were shown by NMR chemical shift perturbation studies through mutagenesis to be important for maintaining a stable active site structure, indicating they are also essential for protein stability [17,86].…”