Biochemical Convergence of Mitochondrial Hsp70 System Specialized in Iron–Sulfur Cluster Biogenesis

Kleczewska, Malgorzata; Grabinska, Aneta; Jelen, Marcin; Stolarska, Milena; Schilke, Brenda; Marszałek, Jarosław; Craig, Elizabeth A.; Dutkiewicz, Rafał

doi:10.3390/ijms21093326

Cited by 16 publications

(15 citation statements)

References 51 publications

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“…HscAs seem to be highly specialized to chaperoning the transfer of Fe-S-clusters from IscU to an apo-enzyme in cooperation with the JDP HscB [for review see ( Puglisi and Pastore, 2018 )]. Whereas HscAs are only found in bacteria, HscB homologs also exist in mitochondria where they either interact with the generalist DnaK-type Hsp70 (in most eukaryotic cells) or with a specialized Hsp70 (some fungi) that emerged by convergent evolution but is still more DnaK-like than HscA-like ( Schilke et al, 2006 ; Kleczewska et al, 2020 ).…”

Section: Hsca and Hscc: Variations Of The Themementioning

confidence: 99%

“…This sequence divergence may have significant mechanistic distinctions but have only been investigated to a limited extent. HscA and HscC are not found outside the prokaryotic kingdom, though, in some fungi, Hsp70s that are specialized for iron sulfur cluster assembly emerged through convergent evolution ( Schilke et al, 2006 ; Kleczewska et al, 2020 ).…”

Section: Introductionmentioning

confidence: 99%

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The Hsp70-Chaperone Machines in Bacteria

Mayer

2021

Front. Mol. Biosci.

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The ATP-dependent Hsp70s are evolutionary conserved molecular chaperones that constitute central hubs of the cellular protein quality surveillance network. None of the other main chaperone families (Tig, GroELS, HtpG, IbpA/B, ClpB) have been assigned with a comparable range of functions. Through a multitude of functions Hsp70s are involved in many cellular control circuits for maintaining protein homeostasis and have been recognized as key factors for cell survival. Three mechanistic properties of Hsp70s are the basis for their high versatility. First, Hsp70s bind to short degenerate sequence motifs within their client proteins. Second, Hsp70 chaperones switch in a nucleotide-controlled manner between a state of low affinity for client proteins and a state of high affinity for clients. Third, Hsp70s are targeted to their clients by a large number of cochaperones of the J-domain protein (JDP) family and the lifetime of the Hsp70-client complex is regulated by nucleotide exchange factors (NEF). In this review I will discuss advances in the understanding of the molecular mechanism of the Hsp70 chaperone machinery focusing mostly on the bacterial Hsp70 DnaK and will compare the two other prokaryotic Hsp70s HscA and HscC with DnaK.

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Section: Hsca and Hscc: Variations Of The Themementioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

The Hsp70-Chaperone Machines in Bacteria

Mayer

2021

Front. Mol. Biosci.

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“…Expression of Ec IscU with a polyhistidine tag at the N-terminus was performed using E. coli strain C41(DE3) carrying pVP67K plasmid encoding IscU-His8 . Expression of Ec IscU was induced by addition of 1 mM IPTG at OD 600nm = 0.6.…”

Section: Methodsmentioning

confidence: 99%

Iron Insertion at the Assembly Site of the ISCU Scaffold Protein Is a Conserved Process Initiating Fe–S Cluster Biosynthesis

et al. 2022

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Iron–sulfur (Fe–S) clusters are prosthetic groups of proteins biosynthesized on scaffold proteins by highly conserved multi-protein machineries. Biosynthesis of Fe–S clusters into the ISCU scaffold protein is initiated by ferrous iron insertion, followed by sulfur acquisition, via a still elusive mechanism. Notably, whether iron initially binds to the ISCU cysteine-rich assembly site or to a cysteine-less auxiliary site via N/O ligands remains unclear. We show here by SEC, circular dichroism (CD), and Mössbauer spectroscopies that iron binds to the assembly site of the monomeric form of prokaryotic and eukaryotic ISCU proteins via either one or two cysteines, referred to the 1-Cys and 2-Cys forms, respectively. The latter predominated at pH 8.0 and correlated with the Fe–S cluster assembly activity, whereas the former increased at a more acidic pH, together with free iron, suggesting that it constitutes an intermediate of the iron insertion process. Iron not binding to the assembly site was non-specifically bound to the aggregated ISCU, ruling out the existence of a structurally defined auxiliary site in ISCU. Characterization of the 2-Cys form by site-directed mutagenesis, CD, NMR, X-ray absorption, Mössbauer, and electron paramagnetic resonance spectroscopies showed that the iron center is coordinated by four strictly conserved amino acids of the assembly site, Cys35, Asp37, Cys61, and His103, in a tetrahedral geometry. The sulfur receptor Cys104 was at a very close distance and apparently bound to the iron center when His103 was missing, which may enable iron-dependent sulfur acquisition. Altogether, these data provide the structural basis to elucidate the Fe–S cluster assembly process and establish that the initiation of Fe–S cluster biosynthesis by insertion of a ferrous iron in the assembly site of ISCU is a conserved mechanism.

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“…De novo synthesis of the [2Fe–2S] cluster is facilitated by Isu1 dimerization induced by a conserved N-terminal tyrosine ( 16 ). In the second stage, target [2Fe–2S] protein assembly is initiated by cluster transfer from Isu1 to the monothiol glutaredoxin Grx5, a reaction assisted by the dedicated Hsp70 chaperone Ssq1 and its J-type cochaperone, Jac1 ( 3 , 17 , 18 , 19 , 20 ). The transiently Grx5-bound [2Fe–2S] cluster is then passed on to target apoproteins without further ISC protein assistance ( 21 , 22 , 23 , 24 ).…”

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confidence: 99%