Mitochondrial Cysteine Desulfurase and ISD11 Coexpressed in <i>Escherichia coli</i> Yield Complex Containing Acyl Carrier Protein

Cai, Kai; Frederick, Ronnie O.; Tonelli, Marco; Markley, John L.

doi:10.1021/acschembio.6b01005

Cited by 34 publications

(42 citation statements)

References 28 publications

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“…We identified recombinant coexpression conditions that allowed the purification of a complex between human NFS1-ISD11 and native Escherichia coli ACP (ACP ec , 44% identical to human mitochondrial ACP) (SI Appendix, Fig. S1), consistent with a recent report (36). An X-ray crystal structure of the NFS1-ISD11-ACP ec (SDA ec ) complex was determined using molecular replacement-single wavelength anomalous dispersion (MR-SAD) and refined to a resolution of 3.09 Å (R work /R free of 21.2/25.9%) with excellent geometry (SI Appendix, Table S1).…”

Section: Resultssupporting

confidence: 87%

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.

145

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: Resultssupporting

confidence: 87%

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.

145

228

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“…3). A similar interaction mode can now be expected for Isd11 attaching Acp1 to the Fe/S cluster biogenesis complex in S. cerevisiae (32,44). The dual role of Acp1 in Fe/S protein biogenesis and mitochondrial fatty acid metabolism, including lipoic acid synthesis, may provide a regulatory device linking the biogenesis of respiratory functions and metabolic activities.…”

Section: De Novo Synthesis Of a [2fe-2s] Cluster On The Mitochondrialsupporting

confidence: 57%

Iron–sulfur cluster biogenesis and trafficking in mitochondria

Braymer

Lill

2017

Journal of Biological Chemistry

320

293

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The biogenesis of iron-sulfur (Fe/S) proteins in eukaryotes is a multistage, multicompartment process that is essential for a broad range of cellular functions, including genome maintenance, protein translation, energy conversion, and the antiviral response. Genetic and cell biological studies over almost 2 decades have revealed some 30 proteins involved in the synthesis of cellular [2Fe-2S] and [4Fe-4S] clusters and their incorporation into numerous apoproteins. Mechanistic aspects of Fe/S protein biogenesis continue to be elucidated by biochemical and ultrastructural investigations. Here, we review recent developments in the pursuit of constructing a comprehensive model of Fe/S protein assembly in the mitochondrion. Ubiquitous iron-sulfur clusters and their synthesis: an overviewIron-sulfur (Fe/S) 2 clusters are inorganic cofactors that are essential for the proper functioning of virtually all biological cells (1). The chemical versatility of these clusters is utilized in fundamental life processes such as energy production, metabolic conversions, DNA maintenance, gene expression regulation, protein translation, and the antiviral response ( Fig. 1) (2-4). In eukaryotes, Fe/S proteins are found in or associated with the mitochondrion, endoplasmic reticulum, cytosol, and the nucleus. These cofactors participate in electron transfer reactions, Lewis acid catalysis, transfer of sulfur atoms, or facilitating structural roles (5, 6). Although the activities of some Fe/S proteins are dispensable for cell survival under certain conditions, for example fungal Fe/S enzymes in the metabolism of amino acids, others such as Fe/S proteins involved in DNA maintenance or protein translation are essential for cell viability (Fig. 1). The growing number of diseases that implicate Fe/S proteins or their assembly factors illustrates the essentiality of the various functions of these protein cofactors (7-9). The phenotypes associated with these "Fe/S diseases" and the in vivo work in model systems such as Saccharomyces cerevisiae and human cell culture have led to a mechanistic model of eukaryotic Fe/S protein biogenesis, in which the sequence of events required for proper synthesis and trafficking of Fe/S clusters has been elucidated (2). This model has been invaluable to diagnosing new mitochondrial disorders, and its continued advancement will enhance the ability for early diagnosis (10 -13). The focus of this review will be on the latest developments of the functional and mechanistic aspects that have advanced the model of mitochondrial Fe/S protein biogenesis.Cells typically maintain a strict balance of iron and sulfide ion concentrations due to their damaging redox reactions when present in excess (14, 15). The cell also uses a complex biosynthetic system to ensure that the sometimes redox-sensitive and labile Fe/S clusters are assembled correctly, trafficked to specific target apoproteins, and remain protected during these processes. This cellular control is exemplified by the 18 known "Fe/S cluster assembly" (ISC) proteins...

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“…As noted above, upon purification, this yields a complex containing the holo-form of E. coli acyl carrier protein [13]. We used ITC to quantify the interaction of (NIA) 2 with ISCU and FXN (here indicating its mature form, FXN 81–210 ).…”

Section: Resultsmentioning

confidence: 99%

“…Mitochondrial ACP is an acidic protein well known for its role in mitochondrial fatty acid synthesis (FASII) [11], but its separate role as an essential component of the human cysteine desulfurase complex that catalyzes Fe-S cluster biosynthesis has only recently come to light [12]. The desulfurase complex produced by co-expressing human ISD11 and NFS1 in E. coli cells contains the covalently-bound 4′-phosphopantetheine form of E. coli acyl carrier protein (Acp) [13]. Because this chimeric complex has been found to exhibit cysteine desulfurase activity and to support Fe-S cluster assembly in vitro [14], E. coli Acp appears to substitute for the human mitochondrial ACP.…”

mentioning

confidence: 99%

“…Because this chimeric complex has been found to exhibit cysteine desulfurase activity and to support Fe-S cluster assembly in vitro [14], E. coli Acp appears to substitute for the human mitochondrial ACP. We determined the stoichiometry of the cysteine desulfurase complex as [NFS1] 2 :[ISD11] 2 :[Acp] 2 [13], hence-forth abbreviated as “(NIA) 2 ”. The presence of E. coli Acp and this stoichiometry has been confirmed by recent X-ray structures of (NIA) 2 complexes produced by co-expressing human ISD11 and NFS1 in E. coli cells [15,16].…”

mentioning

confidence: 99%

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

Cai

Frederick

Tonelli

et al. 2018

Journal of Inorganic Biochemistry

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Frataxin (FXN) is involved in mitochondrial iron-sulfur (Fe-S) cluster biogenesis and serves to accelerate Fe-S cluster formation. FXN deficiency is associated with Friedreich ataxia, a neurodegenerative disease. We have used a combination of isothermal titration calorimetry and multinuclear NMR spectroscopy to investigate interactions among the components of the biological machine that carries out the assembly of iron-sulfur clusters in human mitochondria. Our results show that FXN tightly binds a single Fe2+ but not Fe3+. While FXN (with or without bound Fe2+) does not bind the scaffold protein ISCU directly, the two proteins interact mutually when each is bound to the cysteine desulfurase complex ([NFS1]2:[ISD11]2:[Acp]2), abbreviated as (NIA)2, where “N” represents the cysteine desulfurase (NFS1), “I” represents the accessory protein (ISD11), and “A” represents acyl carrier protein (Acp). FXN binds (NIA)2 weakly in the absence of ISCU but more strongly in its presence. Fe2+-FXN binds to the (NIA)2-ISCU2 complex without release of iron. However, upon the addition of both L-cysteine and a reductant (either reduced FDX2 or DTT), Fe2+ is released from FXN as consistent with Fe2+-FXN being the proximal source of iron for Fe-S cluster assembly.

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

Cited by 34 publications

References 28 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

Iron–sulfur cluster biogenesis and trafficking in mitochondria

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

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