DnaJ (Hsp40 Protein) Binding to Folded Substrate Impacts KplE1 Prophage Excision Efficiency

Puvirajesinghe, Tania M.; Elantak, Latifa; Lignon, Sabrina; Franche, Nathalie; Ilbert, Marianne; Ansaldi, Mireille

doi:10.1074/jbc.m111.331462

Cited by 8 publications

(9 citation statements)

References 30 publications

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“…Analysis of the RepE deletion mutants demonstrates that the N-terminal domain is sufficient to form a stable complex, confirmed by the good docking of this domain in the reconstruction of the complex between DnaJ and RepE . According to the three-dimensional reconstruction of the DnaJ-RepE complex and the conformational changes described above, the chaperone promotes a rearrangement at the level of the C-terminal domain of RepE distant from the binding site, as has been shown for other DnaJ substrates (7,34). We demonstrate that the disordered loop encompassing residues 47-57 (L ␣2-␤2 ) at the N-terminal domain of RepE is essential to promote the conformational activation of the substrate.…”

Section: Discussionsupporting

confidence: 64%

Structural Insights into the Chaperone Activity of the 40-kDa Heat Shock Protein DnaJ

Cuéllar

Perales-Calvo

Muga

et al. 2013

Journal of Biological Chemistry

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Background: DnaJ is an Hsp40 molecular chaperone able to bind and remodel native substrates as RepE, the repressor/ activator of plasmid F replication. Results: We describe the structure of DnaJ bound to RepE and two mutants and the resulting protein conformational changes. Conclusion: DnaJ conformational plasticity allows binding of different substrates. Significance: We present the first structure of an Hsp40 chaperone bound to a client.

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Section: Discussionsupporting

confidence: 64%

Structural Insights into the Chaperone Activity of the 40-kDa Heat Shock Protein DnaJ

Cuéllar

Perales-Calvo

Muga

et al. 2013

Journal of Biological Chemistry

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“…As a result, we did not isolate mutants affected in the RDF activity only. In recent studies [20,26], we also designed several mutants by site-directed mutagenesis which were included in Table 1 ( d mark) and were further analyzed together with the mutants generated by random mutagenesis.…”

Section: Resultsmentioning

confidence: 99%

“…+ indicates the activity of the WT torI allele, − refers to a null anti-RR activity; d Mutants obtained by site-directed mutagenesis [20,26]; e Additional residues: SGSSRVDLQACKLGCFGG; f Additional residues: KDPLESTCRHASLAVLADERRFSA.…”

Section: Resultsmentioning

confidence: 99%

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Insights into the Functions of a Prophage Recombination Directionality Factor

Panis

Franche

Méjean

et al. 2012

Viruses

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Recombination directionality factors (RDFs), or excisionases, are essential players of prophage excisive recombination. Despite the essentially catalytic role of the integrase in both integrative and excisive recombination, RDFs are required to direct the reaction towards excision and to prevent re-integration of the prophage genome when entering a lytic cycle. KplE1, HK620 and numerous (pro)phages that integrate at the same site in enterobacteria genomes (such as the argW tRNA gene) all share a highly conserved recombination module. This module comprises the attL and attR recombination sites and the RDF and integrase genes. The KplE1 RDF was named TorI after its initial identification as a negative regulator of the tor operon. However, it was characterized as an essential factor of excisive recombination. In this study, we designed an extensive random mutagenesis protocol of the torI gene and identified key residues involved in both functions of the TorI protein. We show that, in addition to TorI-TorR protein-protein interaction, TorI interacts in solution with the IntS integrase. Moreover, in vitro, TorR and IntS appear to compete for TorI binding. Finally, our mutagenesis results suggest that the C-terminal part of the TorI protein is dedicated to protein-protein interactions with both proteins TorR and IntS.

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“…Figure 9 shows a cartoon rendition of the model. In the wild-type host, DnaJ interacts with the improperly folded N-terminal hydrophilic domain, thus avoiding In support of this notion, there is a precedent for DnaJ acting as a chaperone independently of DnaK-ATPase heat shock activity, in stabilizing a Lys/Arg-rich C-terminal domain of TorI, the recombination directionality factor of the KplE1 cryptic prophage of E. coli (21). In the ΔdnaJ mutant host, compensatory elevations in other, less specific, chaperones would lead to similar folding events, although with slower kinetics in the absence of the specific interaction.…”

Section: Discussionmentioning

confidence: 99%

MS2 Lysis of Escherichia coli Depends on Host Chaperone DnaJ

2017

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The L protein of the single-stranded RNA phage MS2 causes lysis of Escherichia coli without inducing bacteriolytic activity or inhibiting net peptidoglycan (PG) synthesis. To find host genes required for L-mediated lysis, spontaneous Ill (insensitivity to L lysis) mutants were selected as survivors of L expression and shown to have a missense change of the highly conserved proline (P330Q) in the C-terminal domain of DnaJ. In the dnaJ P330Q mutant host, L-mediated lysis is completely blocked at 30°C without affecting the intracellular levels of L. At higher temperatures (37°C and 42°C), both lysis and L accumulation are delayed. The lysis block at 30°C in the dnaJ P330Q mutant was recessive and could be suppressed by L overcomes dnaJ (L odj ) alleles selected for restoration of lysis. All three L odj alleles lack the highly basic N-terminal half of the lysis protein and cause lysis ϳ20 min earlier than full-length L. DnaJ was found to form a complex with full-length L. This complex was abrogated by the P330Q mutation and was absent with the L odj truncations. These results suggest that, in the absence of interaction with DnaJ, the N-terminal domain of L interferes with its ability to bind to its unknown target. The lysis retardation and DnaJ chaperone dependency conferred by the nonessential, highly basic N-terminal domain of L resembles the SlyD chaperone dependency conferred by the highly basic C-terminal domain of the E lysis protein of X174, suggesting a common theme where single-gene lysis can be modulated by host factors influenced by physiological conditions. IMPORTANCE Small single-stranded nucleic acid lytic phages (Microviridae and Leviviridae) lyse their host by expressing a single "protein antibiotic." The protein antibiotics from two out of three prototypic small lytic viruses have been shown to inhibit two different steps in the conserved PG biosynthesis pathway. However, the molecular basis of lysis caused by L, the lysis protein of the third prototypic virus, MS2, is unknown. The significance of our research lies in the identification of DnaJ as a chaperone in the MS2 L lysis pathway and the identification of the minimal lytic domain of MS2 L. Additionally, our research highlights the importance of the highly conserved P330 residue in the C-terminal domain of DnaJ for specific protein interactions.

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DnaJ (Hsp40 Protein) Binding to Folded Substrate Impacts KplE1 Prophage Excision Efficiency

Cited by 8 publications

References 30 publications

Structural Insights into the Chaperone Activity of the 40-kDa Heat Shock Protein DnaJ

Structural Insights into the Chaperone Activity of the 40-kDa Heat Shock Protein DnaJ

Insights into the Functions of a Prophage Recombination Directionality Factor

MS2 Lysis of Escherichia coli Depends on Host Chaperone DnaJ

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