Structural basis of RICs iron donation for iron-sulfur cluster biogenesis

AbstractEscherichia coli YtfE is a di-iron protein, of the widespread RIC family, with capacity to donate iron, which is a crucial component of the biogenesis of the ubiquitous family of iron-sulfur proteins. Herein we identify in E. coli a previously unrecognized link between the YtfE protein and the major bacterial system for iron-sulfur cluster (ISC) assembly. We show that YtfE establishes protein-protein interactions with the scaffold IscU, where the transient cluster is formed, and the cysteine desulfurase IscS. Moreover, we found that promotion by YtfE of the formation of an Fe-S cluster in IscU requires two glutamates, E125 and E159 in YtfE. Both glutamates form part of the entrance of a protein channel in YtfE that links the di-iron centre to the surface. In particular, E125 is crucial for the exit of iron, as a single mutation to leucine closes the channel rendering YtfE inactive for the build-up of Fe-S clusters. Hence, we provide evidence for the key role of RICs as bacterial iron donor proteins involved in the biogenesis of Fe-S clusters.ImportanceThe ubiquitous iron-sulfur proteins require specialized cellular machineries to promote the assembly of the cofactor. These systems include proteins that provide sulfur and iron, and scaffold proteins where the cluster is formed. Although largely studied the nature of the iron donor remains to be fully clarified. In this work, we show that Escherichia coli YtfE, which belongs to the RIC protein family, establishes protein-protein interactions with two of the major proteins of the ISC system, and we reveal the structural characteristics necessary for the exit of iron ions from YtfE. Altogether our results prove that RICs can be considered a family of iron donor proteins involved in the biogenesis of iron-sulfur containing proteins.

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Structural Basis of RICs Iron Donation for Iron-Sulfur Cluster Biogenesis

Frontiers in Microbiology ◽

10.3389/fmicb.2021.670681 ◽

2021 ◽

Vol 12 ◽

Author(s):

Liliana S. O. Silva ◽

Pedro M. Matias ◽

Célia V. Romão ◽

Lígia M. Saraiva

Keyword(s):

Protein Interactions ◽

Structural Basis ◽

Single Mutation ◽

Protein Protein Interactions ◽

Cysteine Desulfurase ◽

Iron Sulfur Cluster ◽

Sulfur Cluster ◽

E Coli ◽

Iron Sulfur ◽

Crucial Component

Escherichia coli YtfE is a di-iron protein of the widespread Repair of Iron Centers proteins (RIC) family that has the capacity to donate iron, which is a crucial component of the biogenesis of the ubiquitous family of iron-sulfur proteins. In this work we identify in E. coli a previously unrecognized link between the YtfE protein and the major bacterial system for iron-sulfur cluster (ISC) assembly. We show that YtfE establishes protein-protein interactions with the scaffold IscU, where the transient cluster is formed, and the cysteine desulfurase IscS. Moreover, we found that promotion by YtfE of the formation of an Fe-S cluster in IscU requires two glutamates, E125 and E159 in YtfE. Both glutamates form part of the entrance of a protein channel in YtfE that links the di-iron center to the surface. In particular, E125 is crucial for the exit of iron, as a single mutation to leucine closes the channel rendering YtfE inactive for the build-up of Fe-S clusters. Hence, we provide evidence for the key role of RICs as bacterial iron donor proteins involved in the biogenesis of Fe-S clusters.

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The conserved CDC motif in the yeast iron regulator Aft2 mediates iron–sulfur cluster exchange and protein–protein interactions with Grx3 and Bol2

JBIC Journal of Biological Inorganic Chemistry ◽

10.1007/s00775-019-01705-x ◽

2019 ◽

Vol 24 (6) ◽

pp. 809-815 ◽

Cited By ~ 9

Author(s):

Haoran Li ◽

Caryn E. Outten

Keyword(s):

Protein Interactions ◽

Protein Protein Interactions ◽

Iron Sulfur Cluster ◽

Sulfur Cluster ◽

Iron Sulfur ◽

Cluster Exchange

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Network of Protein-Protein Interactions among Iron-Sulfur Cluster Assembly Proteins in Escherichia coli1

The Journal of Biochemistry ◽

10.1093/oxfordjournals.jbchem.a003156 ◽

2002 ◽

Vol 131 (5) ◽

pp. 713-719 ◽

Cited By ~ 77

Author(s):

U. Tokumoto ◽

S. Nomura ◽

Y. Minami ◽

H. Mihara ◽

S.-i. Kato ◽

...

Keyword(s):

Protein Interactions ◽

Cluster Assembly ◽

Protein Protein Interactions ◽

Iron Sulfur Cluster ◽

Sulfur Cluster ◽

Iron Sulfur ◽

Assembly Proteins

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New Techniques for Ancient Proteins: Direct Coupling Analysis Applied on Proteins involved in Iron Sulfur Cluster Biogenesis

10.1101/103283 ◽

2017 ◽

Author(s):

Marco Fantini ◽

Duccio Malinverni ◽

Paolo De Los Rios ◽

Annalisa Pastore

Keyword(s):

Protein Interactions ◽

Strong Support ◽

Scaffold Protein ◽

Direct Coupling ◽

Protein Protein Interactions ◽

Coupling Analysis ◽

Iron Sulfur Cluster ◽

Sulfur Cluster ◽

Iron Sulfur ◽

Direct Coupling Analysis

ABSTRACTDirect coupling analysis (DCA) is a powerful tool based on protein evolution and introduced to predict protein fold and protein-protein interactions which has been applied also to the prediction of entire interactomes. We have used DCA to analyse three proteins of the iron-sulfur biogenesis machine, an essential metabolic pathway conserved in all organisms. We show that, although based on a relatively small number of sequences due to its distribution in genomes, we can correctly recapitulate all the features of the fold of the CyaY/frataxin family, a protein involved in the human disease Friedreich’s ataxia. This result gave us confidence in the use of this tool. Application of DCA to the iron-sulfur cluster scaffold protein IscU, which has been suggested to function both as an ordered and a disordered form, allows us to clearly distinguish evolutionary traces of the structured species, suggesting that, if present in the cell, the disordered form has not left any evolutionary imprinting. We observe instead, for the first time, direct indications of how the protein can dimerize head-to-head and bind 4Fe4S clusters. Analysis of the alternative scaffold protein IscA provides strong support to a coordination of the cluster mediated by a dimeric rather than a tetrameric form as previously suggested. Our analysis also suggests the presence in solution of a mixture of monomeric and dimeric species and guide us to the prevalent one. Finally, we used DCA to analyse protein-protein interactions between some of these proteins and discuss the potentialities and the limitations of the method.

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Functional Studies of [FeFe] Hydrogenase Maturation in an Escherichia coli Biosynthetic System

Journal of Bacteriology ◽

10.1128/jb.188.6.2163-2172.2006 ◽

2006 ◽

Vol 188 (6) ◽

pp. 2163-2172 ◽

Cited By ~ 212

Author(s):

Paul W. King ◽

Matthew C. Posewitz ◽

Maria L. Ghirardi ◽

Michael Seibert

Keyword(s):

Escherichia Coli ◽

Catalytic Domain ◽

Expression System ◽

Iron Sulfur Cluster ◽

Sulfur Cluster ◽

E Coli ◽

Functional Studies ◽

Hydrogenase Maturation ◽

Iron Sulfur ◽

And Function

ABSTRACT Maturation of [FeFe] hydrogenases requires the biosynthesis and insertion of the catalytic iron-sulfur cluster, the H cluster. Two radical S-adenosylmethionine (SAM) proteins proposed to function in H cluster biosynthesis, HydEF and HydG, were recently identified in the hydEF-1 mutant of the green alga Chlamydomonas reinhardtii (M. C. Posewitz, P. W. King, S. L. Smolinski, L. Zhang, M. Seibert, and M. L. Ghirardi, J. Biol. Chem. 279:25711-25720, 2004). Previous efforts to study [FeFe] hydrogenase maturation in Escherichia coli by coexpression of C. reinhardtii HydEF and HydG and the HydA1 [FeFe] hydrogenase were hindered by instability of the hydEF and hydG expression clones. A more stable [FeFe] hydrogenase expression system has been achieved in E. coli by cloning and coexpression of hydE, hydF, and hydG from the bacterium Clostridium acetobutylicum. Coexpression of the C. acetobutylicum maturation proteins with various algal and bacterial [FeFe] hydrogenases in E. coli resulted in purified enzymes with specific activities that were similar to those of the enzymes purified from native sources. In the case of structurally complex [FeFe] hydrogenases, maturation of the catalytic sites could occur in the absence of an accessory iron-sulfur cluster domain. Initial investigations of the structure and function of the maturation proteins HydE, HydF, and HydG showed that the highly conserved radical-SAM domains of both HydE and HydG and the GTPase domain of HydF were essential for achieving biosynthesis of active [FeFe] hydrogenases. Together, these results demonstrate that the catalytic domain and a functionally complete set of Hyd maturation proteins are fundamental to achieving biosynthesis of catalytic [FeFe] hydrogenases.

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