Architecture of the Human Mitochondrial Iron-Sulfur Cluster Assembly Machinery

Gakh, Oleksandr; Ranatunga, Wasantha; Smith, Douglas Y.; Ahlgren, Eva Christina; Al-Karadaghi, Salam; Thompson, James R.; Isaya, Grazia

doi:10.1074/jbc.m116.738542

Cited by 24 publications

(45 citation statements)

References 75 publications

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“…S9), which are conducted at submicromolar concentrations similar to Fe-S assembly protein levels measured in mitochondria (59)(60)(61). Additional EM studies of a much larger and functionally distinct oligomeric form of the eukaryotic Fe-S assembly complex are also consistent with an NFS1 quaternary structure different from IscS (62). Another PLP-containing enzyme, ornithine decarboxylase, also uses quaternary structure differences to modulate activity (63).…”

Section: Discussionmentioning

confidence: 91%

Structure of human Fe–S assembly subcomplex reveals unexpected cysteine desulfurase architecture and acyl-ACP–ISD11 interactions

Cory

Vranken

Brignole

et al. 2017

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

142

228

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In eukaryotes, sulfur is mobilized for incorporation into multiple biosynthetic pathways by a cysteine desulfurase complex that consists of a catalytic subunit (NFS1), LYR protein (ISD11), and acyl carrier protein (ACP). This NFS1-ISD11-ACP (SDA) complex forms the core of the iron-sulfur (Fe-S) assembly complex and associates with assembly proteins ISCU2, frataxin (FXN), and ferredoxin to synthesize Fe-S clusters. Here we present crystallographic and electron microscopic structures of the SDA complex coupled to enzyme kinetic and cell-based studies to provide structure-function properties of a mitochondrial cysteine desulfurase. Unlike prokaryotic cysteine desulfurases, the SDA structure adopts an unexpected architecture in which a pair of ISD11 subunits form the dimeric core of the SDA complex, which clarifies the critical role of ISD11 in eukaryotic assemblies. The different quaternary structure results in an incompletely formed substrate channel and solvent-exposed pyridoxal 5′-phosphate cofactor and provides a rationale for the allosteric activator function of FXN in eukaryotic systems. The structure also reveals the 4′-phosphopantetheine-conjugated acyl-group of ACP occupies the hydrophobic core of ISD11, explaining the basis of ACP stabilization. The unexpected architecture for the SDA complex provides a framework for understanding interactions with acceptor proteins for sulfur-containing biosynthetic pathways, elucidating mechanistic details of eukaryotic Fe-S cluster biosynthesis, and clarifying how defects in Fe-S cluster assembly lead to diseases such as Friedreich's ataxia. Moreover, our results support a lock-and-key model in which LYR proteins associate with acyl-ACP as a mechanism for fatty acid biosynthesis to coordinate the expression, Fe-S cofactor maturation, and activity of the respiratory complexes.ron-sulfur (Fe-S) clusters are protein cofactors and are required for critical biological processes such as oxidative respiration, nitrogen fixation, and photosynthesis. The iron-sulfur cluster (ISC) biosynthetic pathway, which is found in most prokaryotes and in the mitochondrial matrix of eukaryotes, is responsible for the synthesis of Fe-S clusters and distribution of these cofactors to the appropriate target proteins. Despite the homology between analogous components of the prokaryotic and eukaryotic ISC pathways, there are key unexplained differences, such as the requirement of the LYR protein ISD11 and acyl carrier protein (ACP) for function in eukaryotic but not prokaryotic ISC systems (1, 2).Mitochondrial LYR proteins are members of a recently identified superfamily that are characterized by their small size (10-22 kDa), high positive charge, invariant Phe residue, and eponymous LeuTyr-Arg (LYR) motif near their N terminus (3). LYR proteins function as subunits or assembly factors for respiratory complexes I, II, III, and V. Human LYRM4, also known as ISD11, is critical for the function of the Fe-S assembly complex (4-9), whereas LYRM8, a key maturation factor for mitochondrial complex II, ...

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Section: Discussionmentioning

confidence: 91%

Structure of human Fe–S assembly subcomplex reveals unexpected cysteine desulfurase architecture and acyl-ACP–ISD11 interactions

Cory

Vranken

Brignole

et al. 2017

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

142

228

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“…1−12 Each of these proteins has proved difficult to prepare recombinantly on its own, and the presence of ISD11 appears to stabilize the structure of NFS1. We recently discovered that His-tagged human ISD11 when overexpressed in E. coli cells pulls down the holo-form of E. coli acyl carrier protein (Acp).…”

mentioning

confidence: 99%

Mitochondrial Cysteine Desulfurase and ISD11 Coexpressed in Escherichia coli Yield Complex Containing Acyl Carrier Protein

et al. 2017

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Mitochondrial cysteine desulfurase is an essential component of the machinery for iron–sulfur cluster biosynthesis. It has been known that human cysteine desulfurase that is catalytically active in vitro can be prepared by overexpressing in Escherichia coli cells two protein components of this system, the cysteine desulfurase protein NFS1 and the auxiliary protein ISD11. We report here that this active preparation contains, in addition, the holo-form of E. coli acyl carrier protein (Acp). We have determined the stoichiometry of the complex to be [Acp]2:[ISD11]2:[NFS1]2. Acyl carrier protein recently has been found to be an essential component of the iron–sulfur protein biosynthesis machinery in mitochondria; thus, because of the activity of [Acp]2:[ISD11]2:[NFS1]2 in supporting iron–sulfur cluster assembly in vitro, it appears that E. coli Acp can substitute for its human homologue.

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“…On the one hand, the short N‐terminal segment of the mature form of FXN comprising residues 81 and 89 (recombinant variant FXN90‐210 lacks this segment) was completely unstructured, as judged by secondary chemical shifts ; the recombinant protein FXN45‐210 (similar to the intermediate form FXN41‐210) was devoid of a periodic structure in the segments 41–81 . In agreement with the latter, the N‐terminal region of the form FXN41‐210 (in this case, FXN was in the context of a multimeric protein assembly that included NFS) exhibited a nonperiodic structure, as judged by electron microscopy results . In this case, only some short segments showed a helical conformation (residues 52–57 and 68–69, PDB ID: ).…”

Section: Resultsmentioning

confidence: 69%

“…It is worth noting that interaction was also mediated by contacts established by numerous residues located in the C‐terminal domain, thus forming a large contact surface, which suggests a cooperative assembly. Interesting information concerning desulfurase activity of the NFS1/ISD11/ISCU/FXN42‐210 protein complex was obtained showing that FXN42‐210 was active .…”

Section: Discussionmentioning

confidence: 99%

“…Interesting information concerning desulfurase activity of the NFS1/ISD11/ISCU/FXN42-210 protein complex was obtained showing that FXN42-210 was active [37]. The fact that His6-TAT-FXN1-210 was also functional as the activator of the desulfurase complex NFS1/ISD11/ISCU suggests that conformational features were preserved in this variant and that the Nterminal part of the protein did not produce steric clashes (in the context of the multiprotein complex) that might have resulted in a significant loss of FXN functionality.…”

Section: Discussionmentioning

confidence: 99%

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Biophysical characterisation of the recombinant human frataxin precursor

et al. 2018

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Friedreich's ataxia is a disease caused by a decrease in the levels of expression or loss of functionality of the mitochondrial protein frataxin ( FXN ). The development of an active and stable recombinant variant of FXN is important for protein replacement therapy. Although valuable data about the mature form FXN 81‐210 has been collected, not enough information is available about the conformation of the frataxin precursor ( FXN 1‐210). We investigated the conformation, stability and function of a recombinant precursor variant (His6‐ TAT ‐ FXN 1‐210), which includes a TAT peptide in the N‐terminal region to assist with transport across cell membranes. His6‐ TAT ‐ FXN 1‐210 was expressed in Escherichia coli and conditions were found for purifying folded protein free of aggregation, oxidation or degradation, even after freezing and thawing. The protein was found to be stable and monomeric, with the N‐terminal stretch (residues 1–89) mostly unstructured and the C‐terminal domain properly folded. The experimental data suggest a complex picture for the folding process of full‐length frataxin in vitro : the presence of the N‐terminal region increased the tendency of FXN to aggregate at high temperatures but this could be avoided by the addition of low concentrations of GdmCl. The purified precursor was translocated through cell membranes. In addition, immune response against His6‐ TAT ‐ FXN 1‐210 was measured, suggesting that the C‐terminal fragment was not immunogenic at the assayed protein concentrations. Finally, the recognition of recombinant FXN by cellular proteins was studied to evaluate its functionality. In this regard, cysteine desulfurase NFS 1/ ISD 11/ ISCU was activated in vitro by His6‐ TAT ‐ FXN 1‐210. Moreover, the results showed that His6‐ TAT ‐ FXN 1‐210 can be ubiquitinated in vitro by the recently identified frataxin E3 ligase RNF 126, in a similar way as the FXN 1‐210, suggesting that the His6‐ TAT extension does not interfere with the ubiquitination machinery.

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Architecture of the Human Mitochondrial Iron-Sulfur Cluster Assembly Machinery

Cited by 24 publications

References 75 publications

Structure of human Fe–S assembly subcomplex reveals unexpected cysteine desulfurase architecture and acyl-ACP–ISD11 interactions

Structure of human Fe–S assembly subcomplex reveals unexpected cysteine desulfurase architecture and acyl-ACP–ISD11 interactions

Mitochondrial Cysteine Desulfurase and ISD11 Coexpressed in Escherichia coli Yield Complex Containing Acyl Carrier Protein

Biophysical characterisation of the recombinant human frataxin precursor

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