Structural insights into chaperone addiction of toxin-antitoxin systems

International audienceSecB chaperones assist protein export by binding both unfolded proteins and the SecA motor. Certain SecB homologs can also control toxin-antitoxin (TA) systems known to modulate bacterial growth in response to stress. In such TA-chaperone (TAC) systems, SecB assists the folding and prevents degradation of the antitoxin, thus facilitating toxin inhibition. Chaperone dependency is conferred by a C-terminal extension in the antitoxin known as chaperone addiction (ChAD) sequence, which makes the antitoxin aggregation-prone and prevents toxin inhibition. Using TAC of Mycobacterium tuberculosis, we present the structure of a SecB-like chaperone bound to its ChAD peptide. We find differences in the binding interfaces when compared to SecB–SecA or SecB-preprotein complexes, and show that the antitoxin can reach a functional form while bound to the chaperone. This work reveals how chaperones can use discrete surface binding regions to accommodate different clients or partners and thereby expand their substrate repertoire and functions

Bacterial Hsp90 mediates the degradation of aggregation-prone Hsp70-Hsp40 substrates preferentially by HslUV proteolysis

Whereas in eukaryotic cells, the Hsp90s are profusely-studied molecular chaperones controlling protein homeostasis together with Hsp70s, in bacteria, the function of Hsp90 (HtpG) and its collaboration with Hsp70 (DnaK) remains unknown. To uncover physiological processes depending on HtpG and DnaK, we performed comparative quantitative proteomic analyses of insoluble and total protein fractions from unstressed wild type E. coli, and from knockout mutants ΔdnaKdnaJ (ΔKJ), ΔhtpG (ΔG) and ΔdnaKdnaJΔhtpG (ΔKJG) and compared their growth rates under heat-stress also with ΔdnaKdnaJΔhslV. Whereas, expectedly, mutant ΔG showed no proteomic differences with wild-type, ΔKJ expressed more chaperones, proteases and ribosomes and dramatically less metabolic and respiratory enzymes. Unexpectedly, we found that ΔKJG showed higher levels of metabolic and respiratory enzymes and both ΔKJG and ΔdnaKdnaJΔhslV grew better at 37 o C than ΔKJ. The results indicate that bacterial Hsp90 mediates the degradation of aggregation-prone Hsp70-Hsp40 substrates, preferably by the HslUV protease. Significance statement: The molecular chaperones Hsp70 and Hsp90 are among the most abundant and well-conserved proteins in all realms of life, forming together the core of the cellular proteostasis network. In eukaryotes, Hsp90 functions in collaboration with Hsp70; we studied this collaboration in E. coli, combining genetic studies with label-free quantitative proteomics in which both protein abundance and protein solubility were quantified. Bacteria lacking Hsp70 (DnaK) and its co-chaperone DnaJ (ΔdnaKdnaJ) grew slower and contained significantly less key metabolic and respiratory enzymes

Multilevel interaction of the DnaK/DnaJ(HSP70/HSP40) stress-responsive chaperone machine with the central metabolism

Networks of molecular chaperones maintain cellular protein homeostasis by acting at nearly every step in the biogenesis of proteins and protein complexes. Herein, we demonstrate that the major chaperone DnaK/HSP70 of the model bacterium Escherichia coli is critical for the proper functioning of the central metabolism and for the cellular response to carbon nutrition changes, either directly or indirectly via the control of the heat-shock response. We identified carbon sources whose utilization was positively or negatively affected by DnaK and isolated several central metabolism genes (among other genes identified in this work) that compensate for the lack of DnaK and/or DnaK/Trigger Factor chaperone functions in vivo. Using carbon sources with specific entry points coupled to NMR analyses of real-time carbon assimilation, metabolic coproducts production and flux rearrangements, we demonstrate that DnaK significantly impacts the hierarchical order of carbon sources utilization, the excretion of main coproducts and the distribution of metabolic fluxes, thus revealing a multilevel interaction of DnaK with the central metabolism

ClpXP-mediated Degradation of the TAC Antitoxin is Neutralized by the SecB-like Chaperone in Mycobacterium tuberculosis

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A Bacteriophage-Encoded J-Domain Protein Interacts with the DnaK/Hsp70 Chaperone and Stabilizes the Heat-Shock Factor σ³² of <em>Escherichia coli</em>

<div><p>The universally conserved J-domain proteins (JDPs) are obligate cochaperone partners of the Hsp70 (DnaK) chaperone. They stimulate Hsp70's ATPase activity, facilitate substrate delivery, and confer specific cellular localization to Hsp70. In this work, we have identified and characterized the first functional JDP protein encoded by a bacteriophage. Specifically, we show that the ORFan gene <em>057w</em> of the T4-related enterobacteriophage RB43 encodes a <em>bona fide</em> JDP protein, named Rki, which specifically interacts with the <em>Escherichia coli</em> host multifunctional DnaK chaperone. However, in sharp contrast with the three known host JDP cochaperones of DnaK encoded by <em>E. coli</em>, Rki does not act as a generic cochaperone <em>in vivo</em> or <em>in vitro</em>. Expression of Rki alone is highly toxic for wild-type <em>E. coli</em>, but toxicity is abolished in the absence of endogenous DnaK or when the conserved J-domain of Rki is mutated. Further <em>in vivo</em> analyses revealed that Rki is expressed early after infection by RB43 and that deletion of the <em>rki</em> gene significantly impairs RB43 proliferation. Furthermore, we show that mutations in the host <em>dnaK</em> gene efficiently suppress the growth phenotype of the RB43 <em>rki</em> deletion mutant, thus indicating that Rki specifically interferes with DnaK cellular function. Finally, we show that the interaction of Rki with the host DnaK chaperone rapidly results in the stabilization of the heat-shock factor σ³², which is normally targeted for degradation by DnaK. The mechanism by which the Rki-dependent stabilization of σ³² facilitates RB43 bacteriophage proliferation is discussed.</p> </div

Rki is transcribed early during bacteriophage infection.

<p>(A) Early promoter mapping. The WebLogo for RB43 early promoter was determined as described in Nolan <i>et al</i>., 2006. The G nucleotide underlined is the putative transcription start site. Putative up elements and the −10 and −35 region are boxed. (B) Northern blot analysis showing transcription of <i>rki</i> and two control genes known to be transcribed in the early (<i>g43</i>) or late (<i>g37.2</i>) phase of infection. (C) Western blot analysis of whole cell extracts prepared from W3110 cells non-infected (−) or infected with RB43 at a MOI ∼10, during 10, 20, 30, or 60 min at 30°C and revealed using an anti-Rki rabbit antibody.</p

Analysis of the various J-domain chimera phenotypes <i>in vivo</i>.

<p>(A) The RB43 genome region containing the ORF057w, adapted from <a href="http://bacteriophage.bioc.tulane.edu/" target="_blank">http://bacteriophage.bioc.tulane.edu/</a>. Genes in black have orthologs in bacteriophage T4. (B) An alignment of the J-domain primary amino acid sequences using ClustalX. Identical residues are shown in black and conserved substitutions in gray. The limits of α-helical secondary structures in the DnaJ J-domain are also shown (black bars). (C) Complementation of the temperature-sensitive phenotype of the bacterial strain W3110 Δ3 (<i>dnaJ cbpA djlA</i> triple mutant) by the various pBAD22-based J-domain chimeras in the presence of 0.01% L-arabinose inducer. Only the origin of the relevant J-domain is shown on top of the figure, the rest of the protein being always that of <i>E. coli</i>'s DnaJ. (D) Complementation assay for bacteriophage λ<i>c</i>I(λ) plaque formation on strain Δ3 using the pBAD22-based J-domain chimeras in the presence of 0.001% L-arabinose at 30°C. The λ<i>c</i>I<i>dnaJ+</i> transducing bacteriophage (λJ+) is shown as a positive control. (E) Complementation for bacterial motility assay showing the radial growth of strain Δ3 expressing the pBAD22-based J-domain chimeras in the presence of 0.001% L-arabinose. (F) The relative steady state level of the various DnaJ chimera constructs expressed in strain W3110 Δ3 at 30°C in the presence of 0.1% L-arabinose, following SDS–PAGE of the extracts and staining with Coomassie blue.</p

Rki toxicity is DnaK-dependent.

<p>(A) Growth of an isogenic set of W3110 derivatives strains expressing the full length bacteriophage-encode JDP on LB ampicillin plates with or without L-arabinose inducer after 18 h incubation at 30°C. The (+) sign indicates no significant difference compared with the empty vector control, (+/−) indicates an approximate efficiency of colony formation of <10⁻², (−) indicates an efficiency of colony formation of <10⁻⁴. (B) The arabinose inducible pBAD22-based full-length Rki and Rki(H38Q) constructs were expressed at 30°C in W3110 wild type strain in the absence (−) or in the presence of 1% of L-arabinose inducer (+). The L-arabinose inducible pBAD33-based full-length Rki constructs were expressed with or without inducer at 30°C in Δ<i>dnaK</i> (Δ<i>dnaK</i>52::Cm^R) strain alone or in the simultaneous presence of either the compatible empty p29SEN vector or the p29SEN-based <i>dnaK</i> gene under the control of its own native promoter. (C) Immunoprecipitation of pBAD24-based Flag-tagged Rki or Rki(H38Q) proteins expressed in <i>E. coli</i> wild-type strain. Cell lysates and eluates (Pull-down Flag) revealed by western blot analyses using anti-Rki, anti-DnaJ and anti-DnaK antibodies.</p

Rki cochaperone functions <i>in vitro</i>.

<p>(A) Stimulation of DnaK ATPase activity under steady state conditions. DnaK(1 µM) and GrpE (0.5 µM) in the absence or in the presence of DnaJ, Rki or Rki(H38Q) at the indicated concentrations. The percentage hydrolyzed ATP/min is plotted as a function of the final DnaJ concentration used in the reaction mix. (B) Refolding chemically-denatured firefly luciferase (125 nM) by DnaK (500 nM) and GrpE (125 nM) in the presence of 125 nM of either DnaJ, Rki or Rki(H38Q) as indicated. The values of luciferase refolding were normalized to the maximal value obtained with that of wild type DnaJ. (C) Luciferase aggregation protection assay. A representative plot of a luciferase aggregation protection assay is shown with chemically-denatured luciferase (1 µM) alone (no JDP), or in the presence of DnaJ (1 µM), Rki (1 µM or 4 µM). Optical densities were measured at 320 nm and the percentage values were normalized to the luciferase aggregation obtained in the absence of added chaperones.</p

Publisher Correction: Structural insights into chaperone addiction of toxin-antitoxin systems

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