Disordered form of the scaffold protein IscU is the substrate for iron-sulfur cluster assembly on cysteine desulfurase

Abstract

The scaffold protein for iron-sulfur cluster assembly, apo-IscU, populates two interconverting conformational states, one disordered (D) and one structured (S) as revealed by extensive NMR assignments. At pH 8 and 25°C, approximately 70% of the protein is S, and the lifetimes of the states are 1.3 s (S) and 0.50 s (D). Zn (II) and Fe(II) each bind and stabilize structured (S-like) states. Single amino acid substitutions at conserved residues were found that shift the equilibrium toward either the S or the D state. Cluster assembly takes place in the complex between IscU and the cysteine desulfurase, IscS, and our NMR studies demonstrate that IscS binds preferentially the D form of apo-IscU. The addition of 10% IscS to IscU was found to greatly increase H/D exchange at protected amides of IscU, to increase the rate of the S → D reaction, and to decrease the rate of the D → S reaction. In the saturated IscU:IscS complex, IscU is largely disordered. In vitro cluster assembly reactions provided evidence for the functional importance of the S ⇆ D equilibrium. IscU variants that favor the S state were found to undergo a lag phase, not observed with the wild type, that delayed cluster assembly; variants that favor the D state were found to assemble less stable clusters at an intermediate rate without the lag. It appears that IscU has evolved to exist in a disordered conformational state that is the initial substrate for the desulfurase and to convert to a structured state that stabilizes the cluster once it is assembled.amino acid sequence effects on protein stability | protein order-disorder transition | two-dimensional exchange spectroscopy | biogenesis of Fe-S clusters I ron-sulfur (Fe-S) clusters, which are among the most ancient and ubiquitous protein prosthetic groups, function in electron transport, enzymatic catalysis, and chemical sensing reactions or as structural units (1). Humans and other higher eukaryotes utilize the ISC (iron-sulfur cluster) system as the essential Fe-S cluster assembly mechanism in mitochondria, and defects in this system have been linked to a large number of human diseases (2). The prokaryotic ISC system has served as a useful model for understanding Fe-S cluster assembly and delivery. The bacterial system utilizes several proteins that have eukaryotic homologs: IscU (scaffold protein), IscS (cysteine desulfurase), HscB (cochaperone), HscA (chaperone), and CyaY (regulation or iron delivery, analog of human frataxin) (1). Interactions among these proteins have been shown to be critical for efficient Fe-S cluster biogenesis (3). The IscU protein acts as a scaffold on which the Fe-S clusters are assembled and from which the clusters are transferred to various apoproteins. IscS is a homodimeric pyridoxyl-5′-phosphatedependent cysteine desulfurase (4). Each IscS subunit binds an IscU molecule and transfers sulfane sulfur generated from the conversion of cysteine to alanine to the cluster ligand cysteines of IscU (5). HscA and HscB, the DnaK-like chaperone and the DnaJ-like cochaperone pro...

Tangled web of interactions among proteins involved in iron–sulfur cluster assembly as unraveled by NMR, SAXS, chemical crosslinking, and functional studies

“…The conformational transition of IscU has been mimicked and further characterized under various conditions. For example, the binding of Zn 2+ [77], Fe 2+ [101], or [2Fe–2S] [78] stabilizes the S-state (Figs. 5A, B).…”

Functional implications of the interaction between HscB and IscU in the biosynthesis of FeS clusters

“…Indeed, the high stability of cluster ligation on holoIscU when present as complex with HscB may minimize losses of cluster atoms when cluster transfer to nascent FeS apoprotein(s) is not immediately required. In the frame of the complicated interplay among proteins involved in FeS cluster biogenesis [37], it may be worth exploring also the possible effects of HscB-induced structural rigidity of apoIscU as for the interaction with those isc-encoded proteins that are involved in delivering cluster atoms, such as the IscS desulfurase [20,32,36].…”

“…Thus, it appears that IscU has evolved to exist in a disordered conformational state that is the initial substrate for the desulfurase and converts to a structured state that stabilizes the cluster once it is assembled. On these bases, it was concluded that the disordered form of IscU is the substrate for cysteine desulfurasemediated assembly of iron-sulfur clusters [20]. It has also been suggested that forms of IscU with different structural stability play alternate roles in their function as scaffolds during FeS biosynthesis [21].…”

Interactions of iron-bound frataxin with ISCU and ferredoxin on the cysteine desulfurase complex leading to Fe-S cluster assembly

“…The canonical mechanism for Fe-S cluster biogenesis involves the pyridoxal phosphate dependent enzyme cysteine desulfurase, L-cysteine as the source of sulfur, an iron delivery protein, a reductant (ferredoxin), and a scaffold protein [5,6]. We have shown that the human scaffold protein ISCU (like its Escherichia coli counterpart IscU [7]) populates two interconverting conformational states: one that is structured (S), and one that is dynamically disordered (D) [8]. Human cysteine desulfurase (NFS1) differs from that of E. coli (IscS) by the requirement for two accessory proteins: ISD11 and mitochondrial acyl carrier protein (ACP).…”