Structure and Dynamics of the Iron−Sulfur Cluster Assembly Scaffold Protein IscU and Its Interaction with the Cochaperone HscB

Kim, Jin Hae; Füzéry, Anna K.; Tonelli, Marco; Ta, Dennis T.; Westler, William M.; Vickery, Larry E.; Markley, John L.

doi:10.1021/bi9002277

Cited by 88 publications

(159 citation statements)

References 32 publications

Supporting

Mentioning

149

Contrasting

Order By: Relevance

“…This result indicated the presence in solution of at least two species, one well folded, the second one being unfolded. The amount of the unfolded species decreased when we started adding zinc in the growth media, supporting the suggestion that this cation stabilizes the fold by compensating for the absence of the cluster and/or of the binding partner IscS [24]. The HSQC spectrum of samples prepared in this way is that of a well folded monomeric protein with well spread sharp resonances (Fig.…”

Section: Iscu Productionsupporting

confidence: 72%

Of the vulnerability of orphan complex proteins: The case study of the E. coli IscU and IscS proteins

Prischi¹,

Pastore²,

Carroni³

et al. 2010

Protein Expression and Purification

View full text Add to dashboard Cite

IscS and IscU, the two central protein components of the iron sulfur cluster assembly machinery, form a complex that is still relatively poorly characterized. In an attempt to standardize the purification of these proteins for structural studies we have developed a protocol to produce them individually in high concentration and purity. We show that IscS is a rather robust protein as long as it is produced in a PLP loaded form and that this co-factor is essential for fold stability and enzyme activity. In contrast to previous evidence, we also propose that, in contrast with previous evidence, IscU is a thermodynamically stable protein with a well defined fold but, when produced in isolation, is a 'complex-orphan protein' that is prone to unfolding if not stabilised by a co-factor or a protein partner. Our work will facilitate further structural and functional studies of these proteins and eventually lead to a better understanding of the whole machinery.

show abstract

Section: Iscu Productionsupporting

confidence: 72%

Of the vulnerability of orphan complex proteins: The case study of the E. coli IscU and IscS proteins

Prischi¹,

Pastore²,

Carroni³

et al. 2010

Protein Expression and Purification

View full text Add to dashboard Cite

show abstract

“…We recently found that apo-IscU (the protein devoid of a metal ion or Fe-S cluster) from Escherichia coli exists in solution, under reducing conditions, as a mixture of two states: one that is structured (S) and one that is dynamically disordered (D) (7). We show here that substitutions of single amino acids at conserved sites can shift the equilibrium toward either the D or the S state.…”

mentioning

confidence: 75%

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.

Self Cite

169

View full text Add to dashboard Cite

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...

show abstract

“…Yeast Pim1, like Lon-type proteases in bacteria and human mitochondria, is able to recognize both unfolded (24) and folded (25) segments as "degrons," depending on the substrate protein. Adding to the challenge in understanding Isu degradation, structural studies of bacterial IscS and IscU, the orthologs of Nfs1 and Isu, respectively, found that their interaction stabilized the structure of IscU in one case and destabilized it in another (17,(26)(27)(28). Detailed in vitro analysis of Isu degradation by Pim1 are needed to identify the Isu degron recognized by Pim1.…”

Section: Discussionmentioning

confidence: 99%

Cysteine desulfurase Nfs1 and Pim1 protease control levels of Isu, the Fe-S cluster biogenesis scaffold

Song

Marszałek

Craig

2012

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

View full text Add to dashboard Cite

Fe-S clusters are critical prosthetic groups for proteins involved in various critical biological processes. Before being transferred to recipient apo-proteins, Fe-S clusters are assembled on the highly conserved scaffold protein Isu, the abundance of which is regulated posttranslationally on disruption of the cluster biogenesis system. Here we report that Isu is degraded by the Lon-type AAA+ ATPase protease of the mitochondrial matrix, Pim1. Nfs1, the cysteine desulfurase responsible for providing sulfur for cluster formation, is required for the increased Isu stability occurring after disruption of cluster formation on or transfer from Isu. Physical interaction between the Isu and Nfs1 proteins, not the enzymatic activity of Nfs1, is the important factor in increased stability. Analysis of several conditions revealed that high Isu levels can be advantageous or disadvantageous, depending on the physiological condition. During the stationary phase, elevated Isu levels were advantageous, resulting in prolonged chronological lifespan. On the other hand, under iron-limiting conditions, high Isu levels were deleterious. Compared with cells expressing normal levels of Isu, such cells grew poorly and exhibited reduced activity of the heme-containing enzyme ferric reductase. Our results suggest that modulation of the degradation of Isu by the Pim1 protease is a regulatory mechanism serving to rapidly help balance the cell's need for critical ironrequiring processes under changing environmental conditions. proteolysis | Hsp70 | aging | Saccharomyces cerevisiae

show abstract

Structure and Dynamics of the Iron−Sulfur Cluster Assembly Scaffold Protein IscU and Its Interaction with the Cochaperone HscB

Cited by 88 publications

References 32 publications

Of the vulnerability of orphan complex proteins: The case study of the E. coli IscU and IscS proteins

Of the vulnerability of orphan complex proteins: The case study of the E. coli IscU and IscS proteins

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

Cysteine desulfurase Nfs1 and Pim1 protease control levels of Isu, the Fe-S cluster biogenesis scaffold

Contact Info

Product

Resources

About