Structural Basis for Fe–S Cluster Assembly and tRNA Thiolation Mediated by IscS Protein–Protein Interactions

Shi, Rong; Proteau, Ariane; Villarroya, Magda; Moukadiri, Ismaïl; Zhang, Linhua; Trempe, Jean‐François; Matte, Allan; Armengod, M.-Eugenia; Cygler, Mirosław

doi:10.1371/journal.pbio.1000354

Cited by 249 publications

(417 citation statements)

References 66 publications

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“…S7) to estimate the mass of the complex in solution. The size determined (102 kDa) is in agreement with the ðIscUÞ 2 ∶ðIscSÞ 2 stoichiometry of the complex and the X-ray structure, which shows one IscU bound to each IscS subunit (8). Given the size of the complex, backbone signals from IscU residues in the binding site with IscS are expected to be exceedingly broad and not visible in the 1 H-15 N spectrum.…”

Section: Resultssupporting

confidence: 77%

“…The solution studies reported here suggest that the differential affinities of the D and S states of IscU play an important role in efficient iron-sulfur cluster assembly. Our finding that apoIscU is largely disordered when bound to IscS differs from recent reports of X-ray (8), NMR (9), and small angle X-ray scattering (8,10) investigations that found the IscU component to be structured within the IscU:IscS complex.…”

contrasting

confidence: 99%

“…S1B). The D state exhibits little evidence of secondary structure, whereas the secondary structural elements of the S state correspond to those found in an NMR structure of the Zn 2þ complex of IscU (11) and in the X-ray structure of the IscU:IscS complex (8). The as- Fig.…”

Section: Resultssupporting

confidence: 55%

“…The structure of the apo-IscU:IscS complex in solution differs profoundly from that observed by X-ray crystallography (8). It appears that the species crystallized was the small fraction of ordered apo-IscU in the complex stabilized by the crystallization cocktail.…”

Section: Discussionmentioning

confidence: 68%

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

Kim

Tonelli²,

Markley³

2011

Proc. Natl. Acad. Sci. U.S.A.

169

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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...

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Section: Resultssupporting

confidence: 77%

contrasting

confidence: 99%

Section: Resultssupporting

confidence: 55%

Section: Discussionmentioning

confidence: 68%

See 2 more Smart Citations

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

Kim

Tonelli²,

Markley³

2011

Proc. Natl. Acad. Sci. U.S.A.

169

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show abstract

“…Sous cette forme, le soufre est activé, mobilisable et non toxique. IscS s'associe avec IscU (iron-sulfur cluster scaffold), une protéine servant de plate-forme d'assemblage pour les centres Fe-S [15,16]. De façon remarquable, la formation et la stabilisation transitoire de ces centres impliquent trois résidus Cys de IscU, mais aussi le résidu Cys catalytique de IscS [17].…”

Section: Biogenèse Des Centres Fe-sunclassified

Du fer et du soufre dans les protéines

Barras

2014

Med Sci (Paris)

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Iron–Sulfur Cluster Biogenesis in Archaea and Bacteria

Chahal

Boyd

Outten

2004

Encyclopedia of Inorganic and Bioinorganic Chemistry

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Iron–sulfur (Fe–S) clusters are key cofactors required for the activity of essential metalloproteins. Biogenesis of Fe–S clusters is carried out by multiple proteins that work together to mobilize iron and sulfide, assemble nascent clusters, and traffic them to target metalloproteins. While the Isc, Nif, and Suf pathways are the most well‐studied Fe–S cluster biogenesis systems, it is clear that a variety of accessory proteins and metabolites cooperate with these systems for cluster assembly. Here, we review the current status of Fe–S cluster biogenesis research in Bacteria and Archaea.

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Structural Basis for Fe–S Cluster Assembly and tRNA Thiolation Mediated by IscS Protein–Protein Interactions

Abstract: Crystal structures reveal how distinct sites on the cysteine desulfurase IscS bind two different sulfur-acceptor proteins, IscU and TusA, to transfer sulfur atoms for iron-sulfur cluster biosynthesis and tRNA thiolation.

Cited by 249 publications

References 66 publications

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

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

Du fer et du soufre dans les protéines

Iron–Sulfur Cluster Biogenesis in Archaea and Bacteria

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