DnaK duplicates and regionally evolves for the increase of proteomic complexity in bacteria

  1. Zhuo Pan
  2. Li Zhuo
  3. Tian-yu Wan
  4. Rui-yun Chen
  5. Yue-zhong Li

Reviewed by

Read the full article

Listed in

Log in to save this article

Abstract

Hsp70 is important for organismic cells to maintain proteostasis and the chaperone protein is duplicated in all eukaryotes and many prokaryotes. Although the functioning mechanism of Hsp70 has been clearly illuminated, the chaperone duplication and functional evolution has been less investigated. DnaK is a highly conserved bacterial Hsp70 family. Here we showed that the dnaK gene is present in 98.9% bacteria and 6.4% bacteria possess duplicated dnaK s; the occurrence and duplication is positively corelated to the increase of proteomic complexity. We identified the interactomes of the two DnaK paralogs in Myxococcus xanthus DK1622, which are mostly nonoverlapped, but both preferring the α&β domain proteins. Consistent with the proteomes, the MxDnaK substrates are both significantly size-larger and pI-higher than that of the single E. coli DnaK. MxDnaK1 is heat-shock inducible, prefers to bind cytosolic proteins, while MxDnaK2 is decreased by heat shock, and is more associated with membrane proteins. The nucleotide binding domain and the substrate binding beta domain are responsible for the significant changes of DnaK substrates, and the former also determines the dimerization of MxDnaK2, but not MxDnaK1. Our work highlights that DnaK is duplicated and regionally evolved for the increase of proteomic complexity in bacteria.

Article activity feed

  1. Review Commons

    Note: This rebuttal was posted by the corresponding author to Review Commons. Content has not been altered except for formatting.

    Learn more at Review Commons

    Reply to the reviewers

    Reviewer #1 (Evidence, reproducibility and clarity):

    1. It is interesting MxDnaK1 seems to prefer cytosolic proteins while Mx-DnaK2 prefers inner membrane proteins. The domain-swapping experiments seem to suggest that the NBD is important for this difference. How NBD is important is not addressed. Is it due to ATP hydrolysis, NBD-SBD interaction, or co-chaperone interactions?

    Answer: Thanks for your comments. We speculate that the co-chaperone interaction might be the key factor contributing to substrate differences. According to the working principle of Hsp70, its functional diversity is largely determined by substrate differences. Co-chaperones, such as JDPs, play a crucial role in this process as they possess the ability to bind substrates and facilitate their targeted delivery. Therefore, much of the functional diversity of the HSP70s is driven by a diverse class of JDPs 1,2. We found that NBD played important roles in cochaperone recognition of MxDnaKs. Additionally, it is generally accepted that the efficiency of ATP hydrolysis does not significantly impact the substrate recognition of Hsp70. Furthermore, if the NBD-SBD interaction is crucial, the substitution of either the NBD or SBDβ domain might result in similar cell phenotypes, as both alterations disrupt the original NBD-SBDβ interaction. We believe the DnaK proteins and their cochaperones both determine the substrate spectrums. We made corresponding modifications in the revised manuscript. (Page22; Line 488-494 in the marked-up manuscript)

    1. About the interactome analysis, since apyrase was added to remove ATP, it's surprising multiple Hsp40s were found in their analysis. Hsp70-Hsp40 interaction is known to require ATP. This may suggest some of the proteins found in their interactome analysis are artifacts. The authors should perform negative controls for their interactome analysis, such as using a control antibody for their CO-IP and analyze any non-specific binding to their resin.

    In addition, since JDPs were pull-down, is it possible some of the substrates identified are actually substrates for JDPs, not binding directly to DnaKs?

    Answer: This is an interesting question. As you correctly noted, the interaction between Hsp70 and Hsp40 requires ATP. In our experiment, we used apyrase to remove ATP in order to promote tight binding of substrate by DnaK. This methodology was initially described by Calloni, G. et al in 20123, and the authors also identified the co-chaperone protein DnaJ, but with a concentration higher than 77% of the interactors. In our opinions, the incomplete removal of ATP could be the underlying cause of this phenomenon.

    We apologize for the undetailed description in Methods. Actually, we implemented negative controls for each MxDnaK in order to eliminate the potential non-specific interactions with Protein A/G beads or antibodies. Specifically, we conducted a CO-IP experiment without the presence of antibodies to assess any non-specific binding to the Protein A/G beads. To further investigate non-specific binding to the antibodies of MxDnaK2 and MxDnaK1, we utilized the mxdnak2-deleted mutant (strain YL2216) and the MxDnaK1 swapping strain with the MxDnaK2 SBDα (strain YL2204), respectively. As the SBDα of MxDnaK1 was employed as antigen to generate antibodies, and YL2204 can’t be recognized by anti-MxDnaK1 (Figure S5). We believe these controls allowed us to evaluate and exclude the non-specific interactions in our CO-IP. We have improved our description in methods. (Page 27; Line 596-607)

    While one of the main functions of JDPs is to interact with unfolded substrates and facilitate their delivery to Hsp70, there may still be substrates that do not directly bind to Hsp70. It’s thus possible that some of the substrates identified only bind to JDPs. We made corresponding modifications in the revised manuscript. (Page 14; Line 290-292)

    1. For Figure S7, the pull-down assay used His6-tagged JDPs. Ni resin is known to bind Hsp70s non-specifically. It's not surprising DnaK showed up in all the pull-down lanes, especially considering how much DnaK was over-expressed. For some pull-down lanes, the amount of DnaK is much more than that of JDPs, further indicating artifact. The author should include negative controls such as JDPs without His6-tag or any irrelevant protein with His6 tag.

    Answer: Thanks for your suggestion. As you and another reviewer pointed out, there were some flaws in the experimental design of the pulldown assay. These include the non-specific binding of Hsp70 proteins to nickel resin, the absence of a negative control without a tag, and the inappropriate selection of the MBP tag. Thus, we employed the nLuc assay as an alternative to the pulldown experiment to validate the interaction between DnaK and JDP (Figure S9). While our manuscript employed nLuc to confirm protein dimerization, it is worth noting that nLuc assay was originally devised for investigating protein interactions 4.

    1. For the proposed dimer formation in Fig. 4C, there are multiple bands above the monomer bands. What are these forms? It seems the majority of the Cys residues that could form disulfide bonds are in the NBD of MxDnaK2 since constructs with MxDnaK2-NBD form some sort of high-MW bands above the monomer. Does MxDnaK1-NBD also contain Cys at the analogous positions? The fact that MxDnaK1 didn't show disulfide-bonded bands doesn't mean it doesn't form dimer. It depends on where the Cys residues are.

    It's nice the authors did Fig. 4D. However, the authors should include a positive control to show how strong the signal is for a true interaction before interpreting their results.

    Answer: Thank you very much for your comments. In at least three independent experiments, we consistently observed two unidentified bands within the molecular weight range of 70-100 kDa during the purification process of His6-MxDnaK2. These bands appeared to be intermediate in size between the monomeric and dimeric forms of His6-MxDnaK2, and disappeared upon DTT treatment. the unidentified band compositions have been confirmed by LC/MS. The upper band included MxDnaK2 (65.3 kDa) and anti-FlhDC factor of E. coli (WP_001300634.1, 27 kDa). In the lower band, we detected the presence of MxDnaK2 and the 50S ribosomal protein L28 of E. coli (WP_000091955.1, 9 kDa). Based on these findings, we conclude that these two additional bands are the result of the interaction between His6-MxDnaK2 and these two E. coli proteins. The related explanations have been added in the legend of Figure 5. (Page 42; Line 938-942)

    We analyzed the presence of Cys in MxDnaK1 and MxDnaK2. The NBD region of MxDnaK2 contains two Cys, located at positions 15 and 319. MxDnaK1-NBD contain a Cys at position of 316, which is the analogous position of 319-Cys of MxDnaK2. The analogous position of 15-Cys of MxDnaK2 is a Val in MxDnaK1, which might be an important factor contributing to the inability of MxDnaK1 to form oligomers.

    Thanks for your suggestion to add the positive control. We re-performed the nLuc assays including a positive control(αSyn). According to the working principle of the nLuc assay, the amount of fluorescent substrate is limited. Therefore, even for proteins that interact with each other, the fluorescence value gradually decreases and reaches a plateau, similar to the negative control. This gradual decline in fluorescence is a significant indicator of protein interaction. In Figure 4D (Figure 5D in the revision version), we only presented the results of the first 20 minutes of detection. The complete two-hour detection results have been added in the supplementary figure (Figure S14).

    1. line 48: "human HSC70 and HSP70 are 85% identical, and the phenotypes of their knockout mutants are different, which is consistent with their largely nonoverlapping substrates" The authors completely ignored that the promoters of HSC70 and HSP70 are very different.

    Answer: This is our carelessness. Yes, HSC70 and HSP70 exhibit distinct expression patterns, which play important roles in their functional diversity. We modified the sentence in the new version (Page 5; Line 58)

    1. Line 69: "The two PRK00290 proteins, not the other Myxococcus Hsp70s, could alternatively compensate the functions of EcDnaK (DnaK of E. coli) for growth." Please add references for this statement.

    Answer: Added, thanks.

    1. line 191: What's the mechanism for DnaK's role in oxidative stress? Is the disulfide bond formation in Fig. 4 related? Does disulfide-bond change the activity of DnaK?

    Answer: Thanks for your pertinent comments. Honestly, we have no idea about the mechanism for MxDnaK2's role in oxidative stress. In our previous studies, we determined that the deletion of mxdnaK2 resulted in a longer lag phase after H2O2 treatment. Here, our aim was to investigate the impact of region swapping on the cellular function of MxDnaK2. In other bacteria, the critical role that DnaK plays in resistance to oxidative stress stems from the pleotropic functions of this chaperone. By shortening the dwelling time that proteins spend in the unfolded state, the DnaK/DnaJ chaperone system minimizes the risk of metal-catalyzed carbonylation of the side chains of proline, lysine, arginine, and threonine residues, but none of them linked to the dimerization characteristic of DnaK 5-7.

    1. Fig. S9 seems redundant.

    Answer: Deleted, thanks.

    1. line 263, "but the NBD exchange was almost equal to the deletion of the gene with respect to phenotypes." But, the mutant has >50% activity in Fig. 3F.

    Answer: We apologize for the confusing description. The “phenotypes” here indicates “cell phenotypes”. What we really tried to say with this sentence is that the NBD swapping strain of either MxDnaK1 or MxDnaK2 presented identical cell phenotypes with the gene-deleted strain. As we have already provided a detailed description of this result earlier, now we consider this sentence to be redundant and have therefore deleted it. (Page 17; Line 355-356)

    1. line 221-226: the logic is not quite clear.

    Answer: We apologize for the confusing description. In M. xanthus DK1622, MxDnaK1 is essential for cell survival, and an insertion of a second copy of mxdnaK1 in the genome is required for deletion of the in-situ gene. Thus, To verify whether the NBD region is required for the essentiality of MxDnaK1, we performed the region swapping of the in situ MxDnaK1 gene in the att::mxdnaK1 mutant (a DK1622 mutant containing a second copy of mxdnaK1 at attB site), and successfully obtained the MxDnaK1 mutant swapped with the MxDnaK2 NBD region. The experiment indicated that the NBD of MxDnaK1 is essential for the cellular functions of the chaperone. We have added the information and modified the sentences in the manuscript. (Page 15; Line 308-319)

    Minor concerns:

    Please check spelling. There are some typos such as "HPPES" in the Methods.

    Answer: Corrected. Many thanks.

    My areas of expertise are protein biochemistry, genetics, and structural biology on heat shock proteins.

    Reviewer #2 (Evidence, reproducibility and clarity):

    Major comments:

    The manuscript is very nice and interesting, although some of the authors' conclusions are perhaps not well supported by their data. For example:

    1. In the pulldown experiments the lack of interaction between 2747-MxDnaK2, 3015-MxDnaK2 and 1145-MxDnaK1 should be shown in order to support the conclusion made in line 197-198,

    Answer: This is our carelessness. As you and another reviewer pointed out, there are some flaws in the experimental design of the pulldown assay. These include the non-specific binding of Hsp70 proteins to nickel resin, the absence of a negative control without a tag, and the inappropriate selection of the MBP tag. Thus, we employed the nLuc assay as an alternative to the pulldown experiment to validate the interaction between DnaK and JDP (including 2747-MxDnaK2, 3015-MxDnaK2 and 1145-MxDnaK1 interaction) (Figure S9). While our manuscript employed nLuc to confirm protein dimerization, it is worth noting that nLuc assay was originally devised for investigating protein interactions 4.

    1. The only evidence that the NBD of MxDnaK1 is essential for bacterial growth is that this mutation couldn´t be obtained in M. xanthus. However, it could be purified in E. coli. Could the authors do some experiments with the M. xanthus strain without the chromosomal MxDnaK1 and then introduce a plasmid with the mutated gene?

    Answer: We apologize for the confusing description. Actually, we determined the NBD is essential not only from the mutation couldn’t be obtained. In M. xanthus DK1622, MxDnaK1 is essential for cell survival, and in-situ deletion of the gene could be obtained after an insertion of a second copy of mxdnaK1 in the genome at the attB site. To verify whether the NBD region is required for the essentiality of MxDnaK1, we performed the region swapping of the in situ MxDnaK1 gene in the att::_mxdnaK_1 mutant (a DK1622 mutant containing a second copy of _mxdnaK_1), and successfully obtained the MxDnaK1 mutant swapped with the MxDnaK2 NBD region. The experiment indicated that the NBD of MxDnaK1 is essential for the cellular functions of the chaperone. We have added the information and modified the sentences in the manuscript. (Page 15; Line 308-319)

    1. All the experiments with purified proteins were done with MxDnaKs bearing His-tags. It doesn't say explicitly its position, but as they employed a pET28A it is likely that the tag is at the N-terminus, which is close to the linker region. As this tag might interfere, it should be removed for the experiments, or at least a control done with the tag removed.

    Answer: We apologize for the lack of detailed description. As you pointed out, the His-tags are located at the N-terminus of DnaKs. The full lengths of MxDnaK1 and MxDnaK2 are 638 and 607 amino acids. The linker regions are located at amino acid positions 381-386 for MxDnaK1 and 387-392 for MxDnaK2. Therefore, we believe that the His-tag is not close to the linker regions. We have included the information in new manuscript. (Page 24; Line 544-546)

    The purified His6-DnaK proteins were employed for holdase activity assays and in vitro dimerization assays. Several previous studies have utilized the same holdase activity assay method with His-tagged DnaK 8,9. We suggested that the His-tag did not interfere with the holdase activity of DnaK. To exclude the influence of His-tag on oligomerization, we conducted a control with the tag removed in the in vitro dimerization assay and the result show no difference (Figure S13).

    1. The authors state that MxDnaK dimerized in vitro with the NBD, and to disrupt the dimer they used 100 mM DTT, which is a very high concentration. As the protein has the His-tag, it should be removed to corroborate that it is not interfering with the dimerization.

    Answer: Thanks for your suggestion. As mentioned above, to exclude the influence of the His-tag on oligomerization, we conducted a control with the tag removed in the in vitro dimerization assay and the result show no difference (Figure S13).

    1. Why were the pulldown experiments done with MBP-MxDnaKs? Can you show a negative control between the MBP and the JDPs to rule out this interaction? It will be more suitable to do the pulldown assays with the purified MxDnaK´s without the His-tags (and the His-tags JDP that were employed).

    Answer: Thanks for your suggestion. As mentioned above, there are some flaws in the experimental design of the pulldown assay. Thus, we employed the nLuc assay as an alternative to the pulldown experiment to validate the interaction between MxDnaKs and JDPs (Figure S9).

    Minor comments:

    • E. coli´s DnaK is only essential in heat shock conditions and for lambda phage cycle. If MxDnaK1 is similar to this Hsp70, why the substitution of its NBD for the NBD MxDnaK2 would be lethal for bacterial growth?

    Answer: Thanks for the comments. As you correctly point out, DnaK is nonessential in E. coli. But in some other bacteria, DnaK also plays an essential role in cell growth for different reasons 10-12. In our previous studies, we determined that MxDnaK1 is essential in M. xanthus DK1622, and the MxDnaK2 is nonessential. In this study, we performed region swapping and found that only the NBD of MxDnaK1 was unreplaceable. In our opinions, the result indicated that NBD play important roles in the functional diversity between MxDnaK1 and MxDnaK2.

    • I think that the writing should be revised and in the supporting information the captions of the figures should include more information.

    Answer: Thanks a lot for the suggestion. We revised the manuscript and added more information in the legends of supplementary figures.

    Reviewer #2 (Significance):

    -General assessment: This is a nice piece of work which would benefit from revision to address the comments above. The authors showed the roles and differences between two DnaK in the same organism. They track these differences to the subdomains of the MxDnaK´s and co-chaperones. It will be interesting for future works to explore more deeply the co-chaperones and their interactions.

    -Advance: I think that this manuscript fills a gap regarding the role of DnaK duplicated in bacterial strains. -Audience: I would say that the audience is broad and includes scientists interested in protein folding and chaperones, as well as myxobacteria.

    1. Rosenzweig, R., Nillegoda, N. B., Mayer, M. P. & Bukau, B. The Hsp70 chaperone network. Nat Rev Mol Cell Biol 20, 665-680, doi:10.1038/s41580-019-0133-3 (2019).
    2. Kampinga, H. H. & Craig, E. A. The HSP70 chaperone machinery: J proteins as drivers of functional specificity. Nat Rev Mol Cell Biol 11, 579-592, doi:10.1038/nrm2941 (2010).
    3. Calloni, G. et al. DnaK functions as a central hub in the E. coli chaperone network. Cell Rep 1, 251-264, doi:10.1016/j.celrep.2011.12.007 (2012).
    4. Dixon, A. S. et al. NanoLuc Complementation Reporter Optimized for Accurate Measurement of Protein Interactions in Cells. ACS Chem Biol 11, 400-408, doi:10.1021/acschembio.5b00753 (2016).
    5. Fredriksson, A., Ballesteros, M., Dukan, S. & Nystrom, T. Defense against protein carbonylation by DnaK/DnaJ and proteases of the heat shock regulon. J Bacteriol 187, 4207-4213, doi:10.1128/JB.187.12.4207-4213.2005 (2005).
    6. Santra, M., Dill, K. A. & de Graff, A. M. R. How Do Chaperones Protect a Cell's Proteins from Oxidative Damage? Cell Syst 6, 743-751 e743, doi:10.1016/j.cels.2018.05.001 (2018).
    7. Fredriksson, A., Ballesteros, M., Dukan, S. & Nystrom, T. Induction of the heat shock regulon in response to increased mistranslation requires oxidative modification of the malformed proteins. Mol Microbiol 59, 350-359, doi:10.1111/j.1365-2958.2005.04947.x (2006).
    8. Chang, L., Thompson, A. D., Ung, P., Carlson, H. A. & Gestwicki, J. E. Mutagenesis reveals the complex relationships between ATPase rate and the chaperone activities of Escherichia coli heat shock protein 70 (Hsp70/DnaK). J Biol Chem 285, 21282-21291, doi:10.1074/jbc.M110.124149 (2010).
    9. Thompson, A. D., Bernard, S. M., Skiniotis, G. & Gestwicki, J. E. Visualization and functional analysis of the oligomeric states of Escherichia coli heat shock protein 70 (Hsp70/DnaK). Cell Stress Chaperones 17, 313-327, doi:10.1007/s12192-011-0307-1 (2012).
    10. Shonhai, A., Boshoff, A. & Blatch, G. L. The structural and functional diversity of Hsp70 proteins from Plasmodium falciparum. Protein Sci 16, 1803-1818, doi:10.1110/ps.072918107 (2007).
    11. Vermeersch, L. et al. On the duration of the microbial lag phase. Curr Genet 65, 721-727, doi:10.1007/s00294-019-00938-2 (2019).
    12. Burkholder, W. F. et al. Mutations in the C-terminal fragment of DnaK affecting peptide binding. Proc Natl Acad Sci U S A 93, 10632-10637, doi:10.1073/pnas.93.20.10632 (1996).
  2. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

    Learn more at Review Commons

    Referee #2

    Evidence, reproducibility and clarity

    Summary: This manuscript describes interesting studies of two paralogues of the E. coli Hsp70, DnaK, from of M. xanthus: MxDnaK1 and MxDnaK2. MxDnaK1 is similar to E. coli DnaK in terms of heat shock response, subcellular localization, etc. while MxDnaK2 is involved with membrane proteins and does not participate in the heat shock response. The interactome of the Mx DnaK´s are larger than that of E. coli DnaK, and their subcellular localization is also different. Regarding the differences between M. xanthus DnaK´s, MxDnaK2 prefers proteins with a higher hydrophobicity score, consistent with its role associated with membrane proteins. The phenotype of diverse mutants with domain swapping showed that the substitution of the NBD of MxDnaK2 for the NBD of MxDnaK1 led to similar phenotypes as the deletion of MxDnaK2 in terms of sporulation and S motility. Consistently, the interactomes of these variants were reduced in number of substrates in comparison with the wild type enzymes. No obvious effect was observed when the SBD´s subdomains were swept. Both MxDnaK interact with JDPs and NEF cochaperones. However, MxDnaK2 interacts only with one of the NEFs, and it depends on the NBD, and has one specific JDP, whichdepends on the beta-subdomain of the SBD (no information provided regarding NBD). MxDnaK1 interacts with both NEFs and has two specific JDPs, which also seems to depend on the beta subdomain of the SBD. Finally, a phylogenetic analysis reveals that the duplication of the dnak gene in Mx is correlated with the complexity of the proteome.

    Major comments:

    • The manuscript is very nice and interesting, although some of the authors' conclusions are perhaps not well supported by their data. For example: 1) In the pulldown experiments the lack of interaction between 2747-MxDnaK2, 3015-MxDnaK2 and 1145-MxDnaK1 should be shown in order to support the conclusion made in line 197-198, 2) The only evidence that the NBD of MxDnaK1 is essential for bacterial growth is that this mutation couldn´t be obtained in M. xanthus. However, it could be purified in E. coli. Could the authors do some experiments with the M. xanthus strain without the chromosomal MxDnaK1 and then introduce a plasmid with the mutated gene?
    • All the experiments with purified proteins were done with MxDnaKs bearing His-tags. It doesn't say explicitly its position, but as they employed a pET28A it is likely that the tag is at the N-terminus, which is close to the linker region. As this tag might interfere, it should be removed for the experiments, or at least a control done with the tag removed.
    • The authors state that MxDnaK dimerized in vitro with the NBD, and to disrupt the dimer they used 100 mM DTT, which is a very high concentration. As the protein has the His-tag, it should be removed to corroborate that it is not interfering with the dimerization.
    • Why were the pulldown experiments done with MBP-MxDnaKs? Can you show a negative control between the MBP and the JDPs to rule out this interaction? It will be more suitable to do the pulldown assays with the purified MxDnaK´s without the His-tags (and the His-tags JDP that were employed).

    Minor comments:

    • E. coli´s DnaK is only essential in heat shock conditions and for lambda phage cycle. If MxDnaK1 is similar to this Hsp70, why the substitution of its NBD for the NBD MxDnaK2 would be lethal for bacterial growth?
    • I think that the writing should be revised and in the supporting information the captions of the figures should include more information.

    Significance

    General assessment: This is a nice piece of work which would benefit from revision to address the comments above. The authors showed the roles and differences between two DnaK in the same organism. They track these differences to the subdomains of the MxDnaK´s and co-chaperones. It will be interesting for future works to explore more deeply the co-chaperones and their interactions.

    Advance: I think that this manuscript fills a gap regarding the role of DnaK duplicated in bacterial strains.

    Audience: I would say that the audience is broad and includes scientists interested in protein folding and chaperones, as well as myxobacteria.

  3. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

    Learn more at Review Commons

    Referee #1

    Evidence, reproducibility and clarity

    In this study, Pan et al. characterized two Hsp70 DnaKs from Myxococcus xanthus DK1622. Through determining interactomes, the authors defined the differences and similarities between these two DnaKs in interacting with co-chaperones and substrates. Using domain-swapping, the authors analyzed the domain requirements for their functions. Lastly, their bioinformatics analyses seem to suggest the presence of these two DnaKs (i.e., DnaK duplication) is due to the increase of proteomic complexity. Overall, the results are interesting although not surprising. As the authors pointed out, many organisms have multiple Hsp70s with different but overlapping functions. Although multiple experimental approaches were used, the manuscript is generally descriptive without revealing any major mechanistic insights.

    1. It is interesting MxDnaK1 seems to prefer cytosolic proteins while Mx-DnaK2 prefers inner membrane proteins. The domain-swapping experiments seem to suggest that the NBD is important for this difference. How NBD is important is not addressed. Is it due to ATP hydrolysis, NBD-SBD interaction, or co-chaperone interactions?
    2. About the interactome analysis, since apyrase was added to remove ATP, it's surprising multiple Hsp40s were found in their analysis. Hsp70-Hsp40 interaction is known to require ATP. This may suggest some of the proteins found in their interactome analysis are artifacts. The authors should perform negative controls for their interactome analysis, such as using a control antibody for their CO-IP and analyze any non-specific binding to their resin.
      In addition, since JDPs were pull-down, is it possible some of the substrates identified are actually substrates for JDPs, not binding directly to DnaKs?
    3. For Figure S7, the pull-down assay used His6-tagged JDPs. Ni resin is known to bind Hsp70s non-specifically. It's not surprising DnaK showed up in all the pull-down lanes, especially considering how much DnaK was over-expressed. For some pull-down lanes, the amount of DnaK is much more than that of JDPs, further indicating artifact. The author should include negative controls such as JDPs without His6-tag or any irrelevant protein with His6 tag.
    4. For the proposed dimer formation in Fig. 4C, there are multiple bands above the monomer bands. What are these forms? It seems the majority of the Cys residues that could form disulfide bonds are in the NBD of MxDnaK2 since constructs with MxDnaK2-NBD form some sort of high-MW bands above the monomer. Does MxDnaK1-NBD also contain Cys at the analogous positions? The fact that MxDnaK1 didn't show disulfide-bonded bands doesn't mean it doesn't form dimer. It depends on where the Cys residues are.
      It's nice the authors did Fig. 4D. However, the authors should include a positive control to show how strong the signal is for a true interaction before interpreting their results.
    5. line 48: "human HSC70 and HSP70 are 85% identical, and the phenotypes of their knockout mutants are different, which is consistent with their largely nonoverlapping substrates." The authors completely ignored that the promoters of HSC70 and HSP70 are very different.
    6. Line 69: "The two PRK00290 proteins, not the other Myxococcus Hsp70s, could alternatively compensate the functions of EcDnaK (DnaK of E. coli) for growth." Please add references for this statement.
    7. line 191: What's the mechanism for DnaK's role in oxidative stress? Is the disulfide bond formation in Fig. 4 related? Does disulfide-bond change the activity of DnaK?
    8. Fig. S9 seems redundant.
    9. line 263, "but the NBD exchange was almost equal to the deletion of the gene with respect to phenotypes." But, the mutant has >50% activity in Fig. 3F.
    10. line 221-226: the logic is not quite clear.

    Minor concerns:

    Please check spelling. There are some typos such as "HPPES" in the Methods.

    Significance

    In this study, Pan et al. characterized two Hsp70 DnaKs from Myxococcus xanthus DK1622. Through determining interactomes, the authors defined the differences and similarities between these two DnaKs in interacting with co-chaperones and substrates. Using domain-swapping, the authors analyzed the domain requirements for their functions. Lastly, their bioinformatics analyses seem to suggest the presence of these two DnaKs (i.e., DnaK duplication) is due to the increase of proteomic complexity. Overall, the results are interesting although not surprising. As the authors pointed out, many organisms have multiple Hsp70s with different but overlapping functions. Although multiple experimental approaches were used, the manuscript is generally descriptive without revealing any major mechanistic insights.

    My areas of expertise are protein biochemistry, genetics, and structural biology on heat shock proteins.

  4. Version published to 10.1101/2023.06.19.545549v1 on bioRxiv