The unfolding story of the <i>Escherichia coli</i> Hsp70 DnaK: is DnaK a holdase or an unfoldase?

Slepenkov, Sergey V.; Witt, Stephan N.

doi:10.1046/j.1365-2958.2002.03093.x

Cited by 100 publications

(91 citation statements)

References 45 publications

(70 reference statements)

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“…However, several proteins have been identified that, like IscS, preferentially bind and stabilize a disordered target protein (20)(21)(22). Many of these are chaperone proteins that rescue misfolded proteins (23)(24)(25) or are proteins involved in protein degradation (26)(27)(28)(29)(30)(31)(32).…”

Section: Discussionmentioning

confidence: 99%

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.

169

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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...

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Section: Discussionmentioning

confidence: 99%

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.

169

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“…To test this, we utilized the E. coli chaperone DnaK. DnaK was purified in the absence of its co-chaperones DnaJ (the DnaK hydrolysis factor) and GrpE (the nucleotide exchange factor) in an ADP-bound state, also referred to as the high affinity state (17)(18)(19)(20)(21). In this form, DnaK binds proteins that expose hydrophobic surface patches.…”

Section: Volume 287 • Number 45 • November 2 2012mentioning

confidence: 99%

Avirulence Protein 3a (AVR3a) from the Potato Pathogen Phytophthora infestans Forms Homodimers through Its Predicted Translocation Region and Does Not Specifically Bind Phospholipids

Wawra

Agacan

Boddey

et al. 2012

Journal of Biological Chemistry

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Background: AVR3a is a Phytophthora infestans effector that translocates into potato cells dependent on the N-terminal RxLR leader. Results: AVR3a dimerization is inhibited by a mutation that also impairs translocation. Conclusion: Phospholipid binding of AVR3a is probably physiologically irrelevant because only denatured protein molecules bind. Significance: Our findings will help us to understand how oomycete RxLR effectors function.

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“…In addition, several proteins involved in molecular chaperoning (DnaK) (Slepenkov & Witt, 2002), acid tolerance response (YfiD) (Stancik et al, 2002), LPS core region biosynthesis (RfaE) (Valvano et al, 2000), transcriptional regulation and other functions were identified. Interestingly, YbeJ, a putative glutamate/aspartate periplasmic binding and transport protein (Tremoulet et al, 2002) was represented by one of the five unique spots from the wild-type.…”

Section: Proteins Regulated By Yjjqmentioning

confidence: 99%

Characterization of a yjjQ mutant of avian pathogenic Escherichia coli (APEC)

Ewers

Laturnus

et al. 2008

Microbiology

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Infections with extraintestinal avian pathogenic Escherichia coli (APEC) cause significant economic losses in the poultry industry worldwide. In a previous study we applied signature-tagged transposon mutagenesis and identified 28 virulence-associated genes in APEC strain IMT5155 (O2 : H5 : K1). One of them, yjjQ, encodes a putative transcriptional regulator whose function and role in pathogenesis are still unknown. In the present study, this mutant has been characterized. The yjjQ-defective mutant of IMT5155 (M18E10) was out-competed by the wild-type strain in vivo, and infection of chickens with this yjjQ mutant led to strongly reduced bacterial loads in several organs. Expression studies showed that transcription of yjjQ was significantly upregulated in M9 minimal medium. Correspondingly, the yjjQ mutant showed significantly reduced growth in M9 medium. Although the mutation could not be complemented, a yjjQ deletion mutant showed phenotypes similar to the transposon-generated mutant M18E10, whereas deletion and overexpression of the downstream gene bglJ did not cause a growth defect in M9. To identify virulence genes regulated by YjjQ, one-and two-dimensional protein gel electrophoresis was performed. The proteomic analysis revealed that in the yjjQ mutant M18E10 the expression of several genes involved in iron uptake was downregulated and some other genes were upregulated. The regulation of genes involved in iron uptake was shown to occur at the transcription level using real-time RT-PCR. Taking the results together, this functional analysis strongly suggests that YjjQ is a regulator involved in virulence of APEC by affecting iron uptake.

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The unfolding story of the Escherichia coli Hsp70 DnaK: is DnaK a holdase or an unfoldase?

Cited by 100 publications

References 45 publications

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

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

Avirulence Protein 3a (AVR3a) from the Potato Pathogen Phytophthora infestans Forms Homodimers through Its Predicted Translocation Region and Does Not Specifically Bind Phospholipids

Characterization of a yjjQ mutant of avian pathogenic Escherichia coli (APEC)

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