Escherichia coli Thioredoxin-like Protein YbbN Contains an Atypical Tetratricopeptide Repeat Motif and Is a Negative Regulator of GroEL

Lin, Jiusheng; Wilson, Mark A.

doi:10.1074/jbc.m111.238741

Cited by 17 publications

(31 citation statements)

References 48 publications

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“…The involvement of YbbN enzymes with the holdase/chaperone machinery was the first role described to these enzymes. Indeed, EcYbbN co-purifies with GroEL, indicating strong physical interaction between these two proteins [9]. We also observed that XfYbbN co-purified with GroEL from E. coli in nickel affinity chromatography ( Supplementary Fig.…”

Section: Discussionsupporting

confidence: 56%

“…YbbN proteins can present three activities: (i) holdase/co-chaperone; (ii) thiol-disulfide oxidoreductase and (iii) prevention hyperoxidation of target protein by formation of mixed disulfides [9,11,29]. The results described here indicated that the ability of each YbbN enzyme to display such activities depend on the C/S-X-X-C/S motif composition as well as on the presence or absence of a Cys residue homologous to Cys63 of EcYbbN.…”

Section: Discussionmentioning

confidence: 71%

“…The Trx domain of EcYbbN lacks the most N-terminal residue of the typical CXXC motif (SQHC) and as expected this protein displays no oxidoreductase activity [9]. EcYbbN is involved in the regulation of chaperone machinery, possibly by protein-protein physical interactions through its TPR motifs and with the assistance of GroEL [5,6].…”

Section: Introductionmentioning

confidence: 88%

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Investigation on the requirements for YbbN/CnoX displaying thiol-disulfide oxidoreductase and chaperone activities

Meireles

Yokomizo

Netto

2020

Preprint

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YbbN/CnoX are proteins that display a Trx domain linked to a tetratricopeptide (TPR) domain, which are involved in protein-protein interactions and protein folding processes. YbbN from Escherichia coli (EcYbbN) displays a co-chaperone (holdase) activity that is induced by HOCl (bleach). EcYbbN contains a SQHC motif within the Trx domain and displays no thiol-disulfide oxidoreductase activity.EcYbbN also presents a second Cys residue at Trx domain (Cys63) 24 residues away from SQHF motif that can form mixed disulfides with substrates. Here, we compared EcYbbN with two other YbbN proteins: from Xylella fastidiosa (XfYbbN) and from Pseudomonas aeruginosa (PaYbbN). While EcYbbN displays two Cys residues along a SQHC[N24]C motif; XfYbbN and PaYbbN present two and three Cys residues in the CAPC[N24]V and CAPC[N24]C motifs, respectively. These three proteins are representatives of evolutionary conserved YbbN subfamilies. In contrast to EcYbbN, both XfYbbN and PaYbbN: (1) reduced an artificial disulfide (5,5′-dithiobis-(2-nitrobenzoic acid) = DTNB); and (2) supported the peroxidase activity of Peroxiredoxin Q from X. fastidiosa, suggesting that in vivo these proteins might function similarly to the canonical Trx enzymes. Indeed, XfYbbN was reduced by XfTrx reductase with a high catalytic efficiency (kcat/Km=1.27 x 10 7 M -1 .s -1 ), like the canonical XfTrx (XfTsnC). Furthermore, EcYbbN (as described before) and XfYbbN, but not PaYbbN displayed HOCl -induced holdase activity. Remarkably, EcYbbN gained disulfide reductase activity while lost the HOCl-activated chaperone function when the SQHC was replaced by CQHC. In contrast,the XfYbbN C40A mutant lost the disulfide reductase activity, while kept its HOCl-induced chaperone function. Finally, we generated a P. aeruginosa strain with the ybbN gene deleted, which did not present increased sensitivity to heat shock or to oxidants or to reductants. Altogether, our results suggest that different YbbN/CnoX proteins display distinct properties and activities, depending on the presence of the three conserved Cys residues. Graphical AbstractAbbreviations TPR, tetratricopeptide repeat; NADPH, nicotinamide adenine dinucleotide phosphate; Trx, thioredoxin; DTNB, 5,5-dithio-bis-(2-nitrobenzoic acid). KeywordsThioredoxin, tetratricopeptide-repeat domain, oxidoreductase activity, chaperone/holdase activity. S C C C C C Highlights-CXXC motif is required for the thiol-disulfide reductase activity of YbbN proteins. -XfYbbN and PaYbbN display thiol-disulfide oxidoreductase activity -The affinities of XfTrxR for XfYbbN and XfTsnC (canonical Trx) are comparable -XfYbbN and EcYbbN, but not PaYbbN, display holdase activity induced by hypochlorous acid -Engineering EcYbbN/CnoX by inserting a Cys residue in the SQHC motif resulted in a gain of function (thiol-disulfide oxidoreductase activity) and abolished the HOCl-induced holdase activity.

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Section: Discussionsupporting

confidence: 56%

Section: Discussionmentioning

confidence: 71%

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Investigation on the requirements for YbbN/CnoX displaying thiol-disulfide oxidoreductase and chaperone activities

Meireles

Yokomizo

Netto

2020

Preprint

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“…Intrinsically disordered regions (IDRs) can act as scaffolds for larger molecular assemblies. Interestingly, E. coli YbbN has been identified as a DnaK-interacting chaperone with an atypical TPR domain (47,48), and DnaK cooperates with the E. coli Hsp90, ans HtpG (49), in possible analogy to assemblies with eukaryotic cytoplasmic Hsp70s. Nonetheless, a C-terminal defect is manifested in a simple reconstituted refolding assay with only DnaJ and GrpE present, and we observe no deficiency in assays that probe functional interactions between DnaK and these co-chaperones upon C-terminal mutation.…”

Section: Discussionmentioning

confidence: 99%

Conserved, Disordered C Terminus of DnaK Enhances Cellular Survival upon Stress and DnaK in Vitro Chaperone Activity

Smock

Blackburn

Gierasch

2011

Journal of Biological Chemistry

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The 70-kDa heat shock proteins (Hsp70s) function as molecular chaperones through the allosteric coupling of their nucleotide-and substrate-binding domains, the structures of which are highly conserved. In contrast, the roles of the poorly structured, variable length C-terminal regions present on Hsp70s remain unclear. In many eukaryotic Hsp70s, the extreme C-terminal EEVD tetrapeptide sequence associates with co-chaperones via binding to tetratricopeptide repeat domains. It is not known whether this is the only function for this region in eukaryotic Hsp70s and what roles this region performs in Hsp70s that do not form complexes with tetratricopeptide repeat domains. We compared C-terminal sequences of 730 Hsp70 family members and identified a novel conservation pattern in a diverse subset of 165 bacterial and organellar Hsp70s. Mutation of conserved C-terminal sequence in DnaK, the predominant Hsp70 in Escherichia coli, results in significant impairment of its protein refolding activity in vitro without affecting interdomain allostery, interaction with co-chaperones DnaJ and GrpE, or the binding of a peptide substrate, defying classical explanations for the chaperoning mechanism of Hsp70. Moreover, mutation of specific conserved sites within the DnaK C terminus reduces the capacity of the cell to withstand stresses on protein folding caused by elevated temperature or the absence of other chaperones. These features of the C-terminal region support a model in which it acts as a disordered tether linked to a conserved, weak substrate-binding motif and that this enhances chaperone function by transiently interacting with folding clients.The ubiquitously distributed Hsp70 family of molecular chaperones shepherd newly synthesized polypeptide chains, protect cells from stress-induced protein aggregation, assist in protein translocation across membranes, and regulate assembly and disassembly of macromolecular complexes. These physiological functions are accomplished by a two-domain allosteric mechanism in which cycles of ATP binding and hydrolysis in the N-terminal nucleotide-binding domain (NBD) 2 control the binding and release of hydrophobic polypeptide segments in the substrate-binding domain (SBD) (1-3). Although we have gained an increasingly detailed picture of this allosteric transition in Hsp70 chaperones and modes of substrate interaction (4 -7), it is striking that there is much less known about the function of the extreme C-terminal unstructured region (Fig. 1). Binding of the C-terminal region of mammalian Hsc70 to substrate was suggested in earlier work (8, 9), and more recently, the C-terminal segment of eukaryotic cytoplasmic Hsp70s was found to contain a conserved tetratricopeptide repeat (TPR) domain interaction motif that mediates binding with Chip, Hip, or Hop co-chaperones that facilitate the assembly of a variety of Hsp70 complexes (3). Even though bacteria lack homologs of these Hsp70-interacting TPR domain proteins, many, including the extensively characterized E. coli Hsp70 DnaK, have a disord...

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“…3), the transcriptional levels of over 10 heat shock genes were higher in RM6607W than in RM6607R (Table 4), including genes encoding the molecular chaperones DnaK (3.0-fold), DnaJ (3.0-fold), HtpG (2.7-fold), CbpA (2.8-fold), GrpE (2.0-fold), GroES (3.7-fold), and GroEL (3.5-fold). Additionally the ybbN gene, which encodes a thioredoxin-like protein that has been shown to enhance the DnaK-DnaJ-GrpE chaperone system but to repress GroESL chaperonin function and ATPase activity in E. coli (40), also was upregulated in RM6607W. yrfH encodes the heat shock protein Hsp15, which plays a role in recycling free 50S subunit to rebuild translation machinery.…”

Section: Figmentioning

confidence: 99%

RcsB Contributes to the Distinct Stress Fitness among Escherichia coli O157:H7 Curli Variants of the 1993 Hamburger-Associated Outbreak Strains

Carter

Parker

Louie

et al. 2012

Appl Environ Microbiol

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Curli are adhesive fimbriae of Enterobactericaeae and are involved in surface attachment, cell aggregation, and biofilm formation. We reported previously that curli-producing (C ؉ ) variants of E. coli O157:H7 (EcO157) were much more acid sensitive than their corresponding curli-deficient (C ؊ ) variants; however, this difference was not linked to the curli fimbriae per se. Here, we investigated the underlying molecular basis of this phenotypic divergence. We identified large deletions in the rcsB gene of C ؉ variants isolated from the 1993 U.S. hamburger-associated outbreak strains. rcsB encodes the response regulator of the RcsCDB two-component signal transduction system, which regulates curli biogenesis negatively but acid resistance positively. Further comparison of stress fitness revealed that C ؉ variants were also significantly more sensitive to heat shock but were resistant to osmotic stress and oxidative damage, similar to C ؊ variants. Transcriptomics analysis uncovered a large number of differentially expressed genes between the curli variants, characterized by enhanced expression in C ؉ variants of genes related to biofilm formation, virulence, catabolic activity, and nutrient uptake but marked decreases in transcription of genes related to various types of stress resistance. Supplying C ؉ variants with a functional rcsB restored resistance to heat shock and acid challenge in cells but blocked curli production, confirming that inactivation of RcsB in C ؉ variants was the basis of fitness segregation within the EcO157 population. This study provides an example of how genome instability of EcO157 promotes intrapopulation diversification, generating subpopulations carrying an array of distinct phenotypes that may confer the pathogen with survival advantages in diverse environments.

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Escherichia coli Thioredoxin-like Protein YbbN Contains an Atypical Tetratricopeptide Repeat Motif and Is a Negative Regulator of GroEL

Cited by 17 publications

References 48 publications

Investigation on the requirements for YbbN/CnoX displaying thiol-disulfide oxidoreductase and chaperone activities

Investigation on the requirements for YbbN/CnoX displaying thiol-disulfide oxidoreductase and chaperone activities

Conserved, Disordered C Terminus of DnaK Enhances Cellular Survival upon Stress and DnaK in Vitro Chaperone Activity

RcsB Contributes to the Distinct Stress Fitness among Escherichia coli O157:H7 Curli Variants of the 1993 Hamburger-Associated Outbreak Strains

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