DnaK Chaperone System Research Articles

Experimental characterization of de novo proteins and their unevolved random-sequence counterparts

De novo gene emergence provides a route for new proteins to be formed from previously non-coding DNA. Proteins born in this way are considered random sequences and typically assumed to lack defined structure. While it remains unclear how likely a de novo protein is to assume a soluble and stable tertiary structure, intersecting evidence from random sequence and de novo-designed proteins suggests that native-like biophysical properties are abundant in sequence space. Taking putative de novo proteins identified in human and fly, we experimentally characterize a library of these sequences to assess their solubility and structure propensity. We compare this library to a set of synthetic random proteins with no evolutionary history. Bioinformatic prediction suggests that de novo proteins may have remarkably similar distributions of biophysical properties to unevolved random sequences of a given length and amino acid composition. However, upon expression in vitro, de novo proteins exhibit moderately higher solubility which is further induced by the DnaK chaperone system. We suggest that while synthetic random sequences are a useful proxy for de novo proteins in terms of structure propensity, de novo proteins may be better integrated in the cellular system than random expectation, given their higher solubility.

Inhibition of Staphylococcus aureus and biofilm formation by the anthelminthic drug, triclabendazole.

Triclabendazole (TBD) has been widely used in the treatment of helminthic infection. The anti-biofilm activity and antibacterial mechanism of TBD against Staphylococcus aureus were not known. Here, the anti-biofilm activity of TBD against clinical S. aureus isolates from China was systematically evaluated. Under TBD pressure, TBD-induced tolerant S. aureus with elevated TBD minimum inhibitory concentration (MIC) was selected in vitro and the genetic mutations between the parental isolates and TBD-induced tolerant derivatives were determined by whole-genome sequencing. TBD could significantly inhibit biofilm formation at sub-inhibitory concentration and disperse mature biofilm of clinical S. aureus isolates. In addition, TBD displayed bactericidal activity against the bacterial cells embedded in the biofilm and showed anti-persisters activity. Proteomic analysis showed that KEGG pathways of ABC transporters and beta-lactam resistance were significantly changed after TBD exposure. Moreover, SAUSA300_RS08395 (molecular chaperone DnaK), SAUSA300_RS11200 (sensor histidine kinase KdpD), SAUSA300_RS06325 (DNA translocase FtsK) were identified as candidate targets of TBD in S. aureus. Overexpression experiments further demonstrated that the elevated transcriptional level of DnaK resulted in S. aureus growth delay after exposure to a sub-MIC concentration of 1/2× MIC TBD. In conclusion, TBD exhibits antibacterial and anti-biofilm activity against S. aureus possibly by targeting the DnaK chaperone system.

Functional Effects of ARV-1502 Analogs Against Bacterial Hsp70 and Implications for Antimicrobial Activity.

The antimicrobial peptide (AMP) ARV-1502 was designed based on naturally occurring short proline-rich AMPs, including pyrrhocoricin and drosocin. Identification of chaperone DnaK as a therapeutic target in Escherichia coli triggered intense research on the ligand-DnaK-interactions using fluorescence polarization and X-ray crystallography to reveal the binding motif and characterize the influence of the chaperone on protein refolding activity, especially in stress situations. In continuation of this research, 182 analogs of ARV-1502 were designed by substituting residues involved in antimicrobial activity against Gram-negative pathogens. The peptides synthesized on solid-phase were examined for their binding to E. coli and S. aureus DnaK providing 15 analogs with improved binding characteristics for at least one DnaK. These 15 analogs were distinguished from the original sequence by their increased hydrophobicity parameters. Additionally, the influence of the entire DnaK chaperone system, including co-chaperones DnaJ and GrpE on refolding and ATPase activity, was investigated. The increasingly hydrophobic peptides showed a stronger inhibitory effect on the refolding activity of E. coli chaperones, reducing protein refolding by up to 64%. However, these more hydrophobic peptides had only a minor effect on the ATPase activity. The most dramatic changes on the ATPase activity involved peptides with aspartate substitutions. Interestingly, these peptides resulted in a 59% reduction of the ATPase activity in the E. coli chaperone system whereas they stimulated the ATPase activity in the S. aureus system up to 220%. Of particular note is the improvement of the antimicrobial activity against S. aureus from originally >128 µg/mL to as low as 16 µg/mL. Only a single analog exhibited improved activity over the original value of 8 µg/mL against E. coli. Overall, the various moderate-throughput screenings established here allowed identifying (un)favored substitutions on 1) DnaK binding, 2) the ATPase activity of DnaK, 3) the refolding activity of DnaK alone or together with co-chaperones, and 4) the antimicrobial activity against both E. coli and S. aureus.

The DnaK Chaperone System Buffers the Fitness Cost of Antibiotic Resistance Mutations in Mycobacteria.

Chaperones aid in protein folding and maintenance of protein integrity. In doing so, they have the unique ability to directly stabilize resistance-conferring amino acid substitutions in drug targets and to counter the stress imparted by these substitutions, thus supporting heritable antimicrobial resistance (AMR). We asked whether chaperones support AMR in Mycobacterium smegmatis, a saprophytic model of Mycobacterium tuberculosis, the causative agent of tuberculosis (TB). We show that DnaK associates with many drug targets and that DnaK associates more with AMR-conferring mutant RNA polymerase (RNAP) than with wild-type RNAP. In addition, frequency-of-resistance (FOR) and fitness studies reveal that the DnaK system of chaperones supports AMR in antimicrobial targets in mycobacteria, including RNAP and the ribosome. These findings highlight chaperones as potential targets for drugs to overcome AMR in mycobacteria, including M. tuberculosis, as well as in other pathogens.IMPORTANCE AMR is a global problem, especially for TB. Here, we show that mycobacterial chaperones support AMR in M. smegmatis, a nonpathogenic model of M. tuberculosis, the causative agent of TB. In particular, the mycobacterial DnaK system of chaperones supports AMR in the antimicrobial targets RNA polymerase and the ribosome. This is the first report showing a role for protein chaperones in mediating AMR in mycobacteria. Given the widespread role of protein chaperones in enabling genomic diversity, we anticipate that our findings can be extended to other microbes.

AAA+ Molecular Chaperone ClpB in Leptospira interrogans: Its Role and Significance in Leptospiral Virulence and Pathogenesis of Leptospirosis.

Bacterial ClpB is an ATP-dependent disaggregase that belongs to the Hsp100/Clp subfamily of the AAA+ ATPases and cooperates with the DnaK chaperone system in the reactivation of aggregated proteins, as well as promotes bacterial survival under adverse environmental conditions, including thermal and oxidative stresses. In addition, extensive evidence indicates that ClpB supports the virulence of numerous bacteria, including pathogenic spirochaete Leptospira interrogans responsible for leptospirosis in animals and humans. However, the specific function of ClpB in leptospiral virulence still remains to be fully elucidated. Interestingly, ClpB was predicted as one of the L. interrogans hub proteins interacting with human proteins, and pathogen–host protein interactions are fundamental for successful invasion of the host immune system by bacteria. The aim of this review is to discuss the most important aspects of ClpB’s function in L. interrogans, including contribution of ClpB to leptospiral virulence and pathogenesis of leptospirosis, a zoonotic disease with a significant impact on public health worldwide.

The Hsp70 Chaperone System Stabilizes a Thermo-sensitive Subproteome in E. coli

Stress-inducible molecular chaperones have essential roles in maintaining protein homeostasis, but the extent to which they affect overall proteome stability remains unclear. Here, we analyze the effects of the DnaK (Hsp70) system on protein stability in Escherichia coli using pulse proteolysis combined with quantitative proteomics. We quantify ∼1,500 soluble proteins and find ∼500 of these to be protease sensitive under normal growth conditions, indicating a high prevalence of conformationally dynamic proteins, forming a metastable subproteome. Acute heat stress results in the unfolding of an additional ∼200 proteins, reflected in the exposure of otherwise buried hydrophobic regions. Overexpression of the DnaK chaperone system markedly stabilizes numerous thermo-sensitive proteins, including multiple ribosomal proteins and large, hetero-oligomeric proteins containing the evolutionarily ancient c.37 fold (P loop nucleoside triphosphate hydrolases). Thus, the Hsp70 system, in addition to its known chaperone functions, has a remarkable capacity to stabilize proteins in their folded states under denaturing stress conditions.

Recognizability of heterologous co-chaperones with Streptococcus intermedius DnaK and Escherichia coli DnaK.

Streptococcus intermedius DnaK complements the temperature-sensitive phenotype of an Escherichia coli dnaK null mutant only when co-chaperones DnaJ and GrpE are co-expressed. Therefore, whether S. intermedius DnaK and E. coli DnaK can recognize heterologous co-chaperones in vitro was examined. Addition of heterologous GrpE to DnaK and DnaJ partially stimulated adenosine triphosphatase (ATPase) activity, and almost completely stimulated luciferase refolding activity. Addition of heterologous DnaJ to GrpE and DnaK also stimulated ATPase activity; however, significant luciferase refolding activity was not observed. Moreover, E. coli DnaJ had a negative effect on the luciferase refolding activity of the S. intermedius DnaK chaperone system. In E. coli chaperone mutants, with the exception of E. coli DnaJ, stronger expression of the heterologous co-chaperones partially or almost completely complemented the temperature-sensitive-phenotype. These results indicate that all heterologous co-chaperones can at least partially recognize DnaK of a distantly related species. A region of the ATPase domain that is present in the DnaK of gram-negative bacteria is absent from the DnaK of gram-positive bacteria. This region is believed to be important for recognition of co-chaperones from gram-negative bacteria. However, insertion of this segment into S. intermedius DnaK failed to increase its ability to recognize E. coli co-chaperones, implying that this region is unnecessary or insufficient for the recognition of E. coli co-chaperones. Thus, our data suggest that a basic structural similarity is conserved among the components of the S. intermedius and E. coli DnaK chaperone systems, allowing weak associations between heterologous components.

Isolation and Identification of Putative Protein Substrates of the AAA+ Molecular Chaperone ClpB from the Pathogenic Spirochaete Leptospira interrogans

Bacterial ClpB is an ATP-dependent Hsp100 chaperone that reactivates aggregated proteins in cooperation with the DnaK chaperone system and promotes survival of bacteria under stress conditions. A large number of publications also indicate that ClpB supports the virulence of bacteria, including a pathogenic spirochaete Leptospira interrogans responsible for leptospirosis in both animals and humans. However, the exact role of ClpB in bacterial pathogenicity remains poorly characterized. It can be assumed that ClpB, due to its role as the molecular chaperone, mediates refolding of essential bacterial proteins, including the known virulence factors, which may become prone to aggregation under infection-induced stresses. In this study, we identified putative substrates of ClpB from L. interrogans (ClpBLi). For this purpose, we used a proteomic approach combining the ClpB-Trap affinity pull-down assays, Liquid chromatography-tandem mass spectrometry (LC-MS-MS/MS), and bioinformatics analyses. Most of the identified proteins were enzymes predominantly associated with major metabolic pathways like the tricarboxylic acid (TCA) cycle, glycolysis–gluconeogenesis and amino acid and fatty acid metabolism. Based on our proteomic study, we suggest that ClpB can support the virulence of L. interrogans by protecting the conformational integrity and catalytic activity of multiple metabolic enzymes, thus maintaining energy homeostasis in pathogen cells.

Two J domains ensure high cochaperone activity of DnaJ, Escherichia coli heat shock protein 40.

Heat shock protein 70 (Hsp70) chaperone systems consist of Hsp70, Hsp40 and a nucleotide-exchange factor and function to help unfolded proteins achieve their native conformations. Typical Hsp40s assume a homodimeric structure and have both chaperone and cochaperone activity. The dimeric structure is critical for chaperone function, whereas the relationship between the dimeric structure and cochaperone function is hardly known. Here, we examined whether two intact protomers are required for cochaperone activity of Hsp40 using an Escherichia coli Hsp70 chaperone system consisting of DnaK, DnaJ and GrpE. The expression systems were generated and two heterodimeric DnaJs that included a mutated protomer lacking cochaperone activity were purified. Normal chaperone activity was demonstrated by assessing aggregation prevention activity using urea-denatured luciferase. The heterodimeric DnaJs were investigated for cochaperone activity by measuring DnaK ATPase activity and the heat-denatured glucose-6-phosphate dehydrogenase refolding activity of the DnaK chaperone system, and they showed reduced cochaperone activity. These results indicate that two intact protomers are required for high cochaperone activity of DnaJ, suggesting that one homodimeric DnaJ molecule promotes the simultaneous binding of multiple DnaK molecules to one substrate molecule, and that this binding mode is required for the efficient folding of denatured proteins.

An essential regulatory function of the DnaK chaperone dictates the decision between proliferation and maintenance in Caulobacter crescentus.

Hsp70 chaperones are well known for their important functions in maintaining protein homeostasis during thermal stress conditions. In many bacteria the Hsp70 homolog DnaK is also required for growth in the absence of stress. The molecular reasons underlying Hsp70 essentiality remain in most cases unclear. Here, we demonstrate that DnaK is essential in the α-proteobacterium Caulobacter crescentus due to its regulatory function in gene expression. Using a suppressor screen we identified mutations that allow growth in the absence of DnaK. All mutations reduced the activity of the heat shock sigma factor σ32, demonstrating that the DnaK-dependent inactivation of σ32 is a growth requirement. While most mutations occurred in the rpoH gene encoding σ32, we also identified mutations affecting σ32 activity or stability in trans, providing important new insight into the regulatory mechanisms controlling σ32 activity. Most notably, we describe a mutation in the ATP dependent protease HslUV that induces rapid degradation of σ32, and a mutation leading to increased levels of the house keeping σ70 that outcompete σ32 for binding to the RNA polymerase. We demonstrate that σ32 inhibits growth and that its unrestrained activity leads to an extensive reprogramming of global gene expression, resulting in upregulation of repair and maintenance functions and downregulation of the growth-promoting functions of protein translation, DNA replication and certain metabolic processes. While this re-allocation from proliferative to maintenance functions could provide an advantage during heat stress, it leads to growth defects under favorable conditions. We conclude that Caulobacter has co-opted the DnaK chaperone system as an essential regulator of gene expression under conditions when its folding activity is dispensable.

Characterization of the molecular chaperone ClpB from the pathogenic spirochaete Leptospira interrogans.

Leptospira interrogans is a spirochaete responsible for leptospirosis in mammals. The molecular mechanisms of the Leptospira virulence remain mostly unknown. Recently, it has been demonstrated that an AAA+ chaperone ClpB (a member of the Hsp100 family) from L. interrogans (ClpBLi) is not only essential for survival of Leptospira under the thermal and oxidative stresses, but also during infection of a host. The aim of this study was to provide further insight into the role of ClpB in the pathogenic spirochaetes and explore its biochemical properties. We found that a non-hydrolysable ATP analogue, ATPγS, but not AMP-PNP induces the formation of ClpBLi hexamers and stabilizes the associated form of the chaperone. ADP also induces structural changes in ClpBLi and promotes its self-assembly, but does not produce full association into the hexamers. We also demonstrated that ClpBLi exhibits a weak ATPase activity that is stimulated by κ-casein and poly-lysine, and may mediate protein disaggregation independently from the DnaK chaperone system. Unexpectedly, the presence of E. coli DnaK/DnaJ/GrpE did not significantly affect the disaggregation activity of ClpBLi and ClpBLi did not substitute for the ClpBEc function in the clpB-null E. coli strain. This result underscores the species-specificity of the ClpB cooperation with the co-chaperones and is most likely due to a loss of interactions between the ClpBLi middle domain and the E. coli DnaK. We also found that ClpBLi interacts more efficiently with the aggregated G6PDH in the presence of ATPγS rather than ATP. Our results indicate that ClpB’s importance during infection might be due to its role as a molecular chaperone involved in reactivation of protein aggregates.

Substrate Discrimination by ClpB and Hsp104.

ClpB of E. coli and yeast Hsp104 are homologous molecular chaperones and members of the AAA+ (ATPases Associated with various cellular Activities) superfamily of ATPases. They are required for thermotolerance and function in disaggregation and reactivation of aggregated proteins that form during severe stress conditions. ClpB and Hsp104 collaborate with the DnaK or Hsp70 chaperone system, respectively, to dissolve protein aggregates both in vivo and in vitro. In yeast, the propagation of prions depends upon Hsp104. Since protein aggregation and amyloid formation are associated with many diseases, including neurodegenerative diseases and cancer, understanding how disaggregases function is important. In this study, we have explored the innate substrate preferences of ClpB and Hsp104 in the absence of the DnaK and Hsp70 chaperone system. The results suggest that substrate specificity is determined by nucleotide binding domain-1.

Do nucleic acids moonlight as molecular chaperones?

Organisms use molecular chaperones to combat the unfolding and aggregation of proteins. While protein chaperones have been widely studied, here we demonstrate that DNA and RNA exhibit potent chaperone activity in vitro. Nucleic acids suppress the aggregation of classic chaperone substrates up to 300-fold more effectively than the protein chaperone GroEL. Additionally, RNA cooperates with the DnaK chaperone system to refold purified luciferase. Our findings reveal a possible new role for nucleic acids within the cell: that nucleic acids directly participate in maintaining proteostasis by preventing protein aggregation.

Examination of ClpB Quaternary Structure and Linkage to Nucleotide Binding

E. coli ClpB is a heat shock protein that belongs to the AAA+ protein family. Studies have shown that ClpB and its eukaryotic homologue, Hsp104, can disaggregate denatured proteins by themselves or cooperate with the DnaK chaperone system in vivo. It is thought that ClpB requires binding of nucleoside triphosphate to assemble into hexameric rings with protein binding activity and ClpB majorly exist as hexamer in the presence of nucleoside triphosphate. In contrast to this conclusion, our sedimentation velocity data show that ClpB resides in a monomer-dimer-tetramer-hexamer equilibrium in the presence of ATPϒS (a slowly hydrolysable ATP analog). ClpB hexamers exhibit fast subunit exchange in the absence of nucleoside triphosphate, while the exchange rates decrease when the binding of nucleotide approaching to saturation. For the first time, we determined the binding constants and stoichiometries for ATPϒS to each ClpB oligomer. The monomer is only able to bind one nucleotide whereas all twelve sites in the hexameric ring are bound by nucleotide. Interestingly, dimers and tetramers exhibit stoichiometries of ∼3 and 7, respectively, which is one fewer than the maximum number of binding sites, which suggest an open conformation for the intermediates. We also determined the assembly constants for dimers, tetramers, and hexamers and their dependencies on nucleotide. These interaction constants make it possible to predict the concentration of hexamers present and able to bind to co-chaperones and polypeptide substrates. We anticipate our studies on ClpB assembly to be a starting point for understanding how ClpB hexamers disaggregate protein aggregates.

Thermodynamic and Hydrodynamic Examination of ClpB Assembly

E. coli ClpB is a heat shock protein that belongs to the AAA+ protein family. Studies have shown that ClpB and its eukaryotic homologue, Hsp104, can disaggregate denatured proteins by themselves or cooperate with the DnaK chaperone system in vivo. It is thought that ClpB requires binding of nucleoside triphosphate to assemble into hexameric rings with protein binding activity and ClpB majorly exist as hexamer in the presence of nucleoside triphosphate. In contrast to this conclusion, our sedimentation velocity data show that ClpB can form hexamer in the absence of nucleotide and ClpB resides in a monomer-dimer-tetramer-hexamer equilibrium in the presence of ATPgS (a slowly hydrolysable ATP analog). ClpB hexamers exhibit fast subunits exchange in the absence of nucleoside triphosphate, while the exchange rates decrease in the presence of a large excess of ATPgS. We anticipate our studies on ClpB assembly to be a starting point of for understanding how ClpB hexamers disaggregate protein aggregates. For example, knowing the population of ClpB hexamers in solution is essential for the interpretation of it is only possible to study the energetics and kinetics data for the of ClpB catalyzed disaggregation processreaction by knowing the population of ClpB hexamer in solution.

Examination of polypeptide substrate specificity for Escherichia coli ClpB.

Escherichia coli ClpB is a molecular chaperone that belongs to the Clp/Hsp100 family of AAA+ proteins. ClpB is able to form a hexameric ring structure to catalyze protein disaggregation with the assistance of the DnaK chaperone system. Our knowledge of the mechanism of how ClpB recognizes its substrates is still limited. In this study, we have quantitatively investigated ClpB binding to a number of unstructured polypeptides using steady-state anisotropy titrations. To precisely determine the binding affinity for the interaction between ClpB hexamers and polypeptide substrates the titration data were subjected to global non-linear least squares analysis incorporating the dynamic equilibrium of ClpB assembly. Our results show that ClpB hexamers bind tightly to unstructured polypeptides with binding affinities in the range of ∼3-16 nM. ClpB exhibits a modest preference of binding to Peptide B1 with a binding affinity of (1.7 ± 0.2) nM. Interestingly, we found that ClpB binds to an unstructured polypeptide substrate of 40 and 50 amino acids containing the SsrA sequence at the C-terminus with an affinity of (12 ± 3) nM and (4 ± 2) nM, respectively. Whereas, ClpB binds the 11-amino acid SsrA sequence with an affinity of (140 ± 20) nM, which is significantly weaker than other polypeptide substrates that we tested here. We hypothesize that ClpB, like ClpA, requires substrates with a minimum length for optimal binding. Finally, we present evidence showing that multiple ClpB hexamers are involved in binding to polypeptides ≥152 amino acids.

The selective roles of chaperone systems on over-expression of human-like collagen in recombinant Escherichia coli.

Human-like collagen (HLC) is a novel biomedical material with promising applications. Usually, insoluble HLC was formed due to over-expression. In order to improve the production of soluble HLC, the effective chaperone proteins and their mediation roles on HLC were clarified. Trigger factor (TF) pathway with low specificity and high binding affinity to nascent chains could increase soluble HLC expression; GroEL-GroES could increase the expression level of HLC by assisting the correct folding of HLC and increase mRNA level of the gene coding for HLC by enhancing mRNA stability. DnaK chaperone system did not work positively on soluble HLC due to the unbalanced ratio of DnaK:DnaJ:GrpE, especially too high GrpE significantly inhibited DnaK-mediated refolding. The production of soluble HLC with co-expression of exogenous TF and GroEL-GroES was increased by 35.3 % in comparison with the highest value 0.26 g/L reported previously.

DnaJ-Promoted Binding of DnaK to Multiple Sites on σ 32 in the Presence of ATP

The Escherichia coli DnaK chaperone system is a canonical heat shock protein 70 (Hsp70) chaperone system comprising Hsp70, Hsp40, and a nucleotide exchange factor. Although Hsp40 is known to facilitate the effective binding of Hsp70 to substrates, the role of Hsp40 in Hsp70-substrate interactions has not yet been fully elucidated. Using the E. coli heat shock transcription factor σ(32) as a substrate in the DnaK chaperone system, we here provide new insight into the Hsp70-substrate interaction. When DnaK-σ(32) complexes formed under various conditions were analyzed by gel filtration, several DnaK-σ(32) complexes with different molecular masses were detected. The results indicated that multiple DnaK molecules simultaneously bind to σ(32), even though it has been suggested that DnaK interacts with σ(32) at a molar ratio of 1:1. Two σ(32) mutants, L201D σ(32) and I54A σ(32), which have reduced affinities for DnaK and DnaJ (Hsp40), respectively, were used to further characterize DnaK-σ(32) complex formation. Pulldown assays demonstrated that the affinity of I54A σ(32) for DnaK was similar to that of wild-type σ(32) in the absence of DnaJ, whereas L201D σ(32) exhibited an extremely low affinity for DnaK. However, in the presence of ATP and DnaJ, the yield of DnaK eluted with L201D σ(32) was much higher than that eluted with I54A σ(32). These results indicate that there are multiple DnaK binding sites on σ(32) and that DnaJ strongly promotes DnaK binding to any site in the presence of ATP, regardless of the intrinsic affinity of DnaK for the site.

Biochemical characterization of the apicoplast-targeted AAA+ ATPase ClpB from Plasmodium falciparum

ClpB is a molecular chaperone from the AAA+ superfamily of ATPases, which reactivates aggregated proteins in cooperation with the DnaK chaperone system. ClpB is essential for infectivity and in-host survival of a number of pathogenic microorganisms, but systematic studies on ClpB from pathogens have not been reported yet. We purified and characterized one of the two ClpB isoforms from the malaria parasite Plasmodium falciparum, PfClpB1. PfClpB1 is targeted to the apicoplast, an essential plastid organelle that is a promising anti-malaria drug target. PfClpB1 contains all characteristic AAA+ sequence motifs, but the middle domain of PfClpB1 includes a 52-residue long non-conserved insert. Like in most AAA+ ATPases, ATP induces self-association of PfClpB1 into hexamers. PfClpB1 catalyzes the hydrolysis of ATP and its ATPase activity is activated in the presence of casein and poly-lysine. Similar to Escherichia coli ClpB, PfClpB1 reactivates aggregated firefly luciferase, but the PfClpB1-mediated aggregate reactivation is inhibited in the presence of E. coli DnaK, DnaJ, and GrpE. The lack of effective cooperation between PfClpB1 and the bacterial DnaK system may arise from the Plasmodium-specific sequence of the ClpB middle domain. Our results indicate that the chaperone activity of PfClpB1 may support survival of Plasmodium falciparum by maintaining the folding status and activity of apicoplast proteins.

Horizontal gene transfer of a chloroplast DnaJ-Fer protein to Thaumarchaeota and the evolutionary history of the DnaK chaperone system in Archaea

BackgroundIn 2004, we discovered an atypical protein in metagenomic data from marine thaumarchaeotal species. This protein, referred as DnaJ-Fer, is composed of a J domain fused to a Ferredoxin (Fer) domain. Surprisingly, the same protein was also found in Viridiplantae (green algae and land plants). Because J domain-containing proteins are known to interact with the major chaperone DnaK/Hsp70, this suggested that a DnaK protein was present in Thaumarchaeota. DnaK/Hsp70, its co-chaperone DnaJ and the nucleotide exchange factor GrpE are involved, among others, in heat shocks and heavy metal cellular stress responses.ResultsUsing phylogenomic approaches we have investigated the evolutionary history of the DnaJ-Fer protein and of interacting proteins DnaK, DnaJ and GrpE in Thaumarchaeota. These proteins have very complex histories, involving several inter-domain horizontal gene transfers (HGTs) to explain the contemporary distribution of these proteins in archaea. These transfers include one from Cyanobacteria to Viridiplantae and one from Viridiplantae to Thaumarchaeota for the DnaJ-Fer protein, as well as independent HGTs from Bacteria to mesophilic archaea for the DnaK/DnaJ/GrpE system, followed by HGTs among mesophilic and thermophilic archaea.ConclusionsWe highlight the chimerical origin of the set of proteins DnaK, DnaJ, GrpE and DnaJ-Fer in Thaumarchaeota and suggest that the HGT of these proteins has played an important role in the adaptation of several archaeal groups to mesophilic and thermophilic environments from hyperthermophilic ancestors. Finally, the evolutionary history of DnaJ-Fer provides information useful for the relative dating of the diversification of Archaeplastida and Thaumarchaeota.