Skip to main content
SpringerLink
Log in

A disulfide-bonded DnaK dimer is maintained in an ATP-bound state

  • Original Paper
  • Published:
Cell Stress and Chaperones Aims and scope

Abstract

DnaK, a major Hsp70 molecular chaperones in Escherichia coli, is a widely used model for studying Hsp70s. We recently solved a crystal structure of DnaK in complex with ATP and showed that DnaK was packed as a dimer in the crystal structure. Our previous biochemical studies supported the formation of a specific DnaK dimer as observed in the crystal structure in solution in the presence of ATP and suggested an important role of this dimer in efficient interaction with Hsp40 co-chaperones. In this study, we dissected the biochemical properties of this DnaK dimer. To restrict DnaK in this dimer form, we mutated two residues on the dimer interface to cysteine, A303C, and H541C. Upon oxidation, this DnaK-A303C-H541C protein formed a specific dimer linked by disulfide bonds formed between A303C and H541C only in the presence of ATP, consistent with the crystal structure. Intriguingly, this disulfide-bond-linked dimer of DnaK-A303C-H541C has reduced ATPase activity and decreased affinity for peptide substrate. More interestingly, unlike wild-type DnaK, the peptide substrate-binding kinetics of this dimer is drastically accelerated even in the absence of ATP, suggesting this dimer is restricted in an ATP-bound conformation regardless of nucleotide bound, which was further supported by our analysis using tryptophan fluorescence and ATP-induced peptide release. Thus, formation of the dimer restricted DnaK in an ATP-bound state and blocked the progression through the chaperone cycle. Productive progression through the chaperone cycle requires the dissociation of this transient dimer. Surprisingly, a significantly compromised interaction with Hsp40 co-chaperone was observed for this disulfide-bond-linked dimer. Thus, dissociation of this DnaK dimer is equally crucial for efficient Hsp40 interaction. An initial interaction between Hsp70 and Hsp40 requires the formation of DnaK dimer; but a stable Hsp70-Hsp40 interaction may follow the dissociation of the dimer.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7

Similar content being viewed by others

References

  • Ahmad A, Bhattacharya A, McDonald RA, Cordes M, Ellington B, Bertelsen EB, Zuiderweg ER (2011) Heat shock protein 70 kDa chaperone/DnaJ cochaperone complex employs an unusual dynamic interface. Proc Natl Acad Sci U S A 108(47):18966–18971

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Alderson TR, Kim JH, Markley JL (2016) Dynamical structures of Hsp70 and Hsp70-Hsp40 complexes. Structure 24(7):1014–1030

    Article  CAS  PubMed  Google Scholar 

  • Balch WE, Morimoto RI, Dillin A, Kelly JW (2008) Adapting proteostasis for disease intervention. Science 319(5865):916–919

    Article  CAS  PubMed  Google Scholar 

  • Benaroudj N, Batelier G, Triniolles F, Ladjimi MM (1995) Self-association of the molecular chaperone HSC70. Biochemistry 34(46):15282–15290

    Article  CAS  PubMed  Google Scholar 

  • Blond-Elguindi S, Cwirla SE, Dower WJ, Lipshutz RJ, Sprang SR, Sambrook JF, Gething MJ (1993) Affinity panning of a library of peptides displayed on bacteriophages reveals the binding specificity of BiP. Cell 75(4):717–728

    Article  CAS  PubMed  Google Scholar 

  • Boorstein WR, Ziegelhoffer T, Craig EA (1994) Molecular evolution of the HSP70 multigene family. J Mol Evol 38(1):1–17

    Article  CAS  PubMed  Google Scholar 

  • Brodsky JL, Chiosis G (2006) Hsp70 molecular chaperones: emerging roles in human disease and identification of small molecule modulators. Curr Top Med Chem 6(11):1215–1225

    Article  CAS  PubMed  Google Scholar 

  • Buchberger A, Theyssen H, Schroder H, McCarty JS, Virgallita G, Milkereit P, Reinstein J, Bukau B (1995) Nucleotide-induced conformational changes in the ATPase and substrate binding domains of the DnaK chaperone provide evidence for interdomain communication. J Biol Chem 270(28):16903–16910

    Article  CAS  PubMed  Google Scholar 

  • Bukau B, Horwich AL (1998) The Hsp70 and Hsp60 chaperone machines. Cell 92(3):351–366

    Article  CAS  PubMed  Google Scholar 

  • Bukau B, Deuerling E, Pfund C, Craig EA (2000) Getting newly synthesized proteins into shape. Cell 101(2):119–122

    Article  CAS  PubMed  Google Scholar 

  • Bukau B, Weissman J, Horwich A (2006) Molecular chaperones and protein quality control. Cell 125(3):443–451

    Article  CAS  PubMed  Google Scholar 

  • Burkholder WF, Zhao X, Zhu X, Hendrickson WA, Gragerov A, Gottesman ME (1996) Mutations in the C-terminal fragment of DnaK affecting peptide binding. Proc Natl Acad Sci U S A 93(20):10632–10637

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Carlino A, Toledo H, Skaleris D, DeLisio R, Weissbach H, Brot N (1992) Interactions of liver Grp78 and Escherichia coli recombinant Grp78 with ATP: multiple species and disaggregation. Proc Natl Acad Sci U S A 89(6):2081–2085

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Clerico EM, Tilitsky JM, Meng W, Gierasch LM (2015) How hsp70 molecular machines interact with their substrates to mediate diverse physiological functions. J Mol Biol 427(7):1575–1588

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Cupp-Vickery JR, Peterson JC, Ta DT, Vickery LE (2004) Crystal structure of the molecular chaperone HscA substrate binding domain complexed with the IscU recognition peptide ELPPVKIHC. J Mol Biol 342(4):1265–1278

    Article  CAS  PubMed  Google Scholar 

  • Davis JE, Voisine C, Craig EA (1999) Intragenic suppressors of Hsp70 mutants: interplay between the ATPase- and peptide-binding domains. Proc Natl Acad Sci U S A 96(16):9269–9276

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Eisenberg E, Greene LE (2007) Multiple roles of auxilin and hsc70 in clathrin-mediated endocytosis. Traffic 8(6):640–646

    Article  CAS  PubMed  Google Scholar 

  • Evans CG, Chang L, Gestwicki JE (2010) Heat shock protein 70 (hsp70) as an emerging drug target. J Med Chem 53(12):4585–4602

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Fan CY, Lee S, Cyr DM (2003) Mechanisms for regulation of Hsp70 function by Hsp40. Cell Stress Chaperones 8(4):309–316

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Flaherty KM, DeLuca-Flaherty C, McKay DB (1990) Three-dimensional structure of the ATPase fragment of a 70 K heat-shock cognate protein. Nature 346(6285):623–628

    Article  CAS  PubMed  Google Scholar 

  • Flynn GC, Chappell TG, Rothman JE (1989) Peptide binding and release by proteins implicated as catalysts of protein assembly. Science 245(4916):385–390

    Article  CAS  PubMed  Google Scholar 

  • Gao B, Eisenberg E, Greene L (1996) Effect of constitutive 70-kDa heat shock protein polymerization on its interaction with protein substrate. J Biol Chem 271(28):16792–16797

    Article  CAS  PubMed  Google Scholar 

  • Gassler CS, Buchberger A, Laufen T, Mayer MP, Schroder H, Valencia A, Bukau B (1998) Mutations in the DnaK chaperone affecting interaction with the DnaJ cochaperone. Proc Natl Acad Sci U S A 95(26):15229–15234

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Gragerov A, Zeng L, Zhao X, Burkholder W, Gottesman ME (1994) Specificity of DnaK-peptide binding. J Mol Biol 235(3):848–854

    Article  CAS  PubMed  Google Scholar 

  • Greene MK, Maskos K, Landry SJ (1998) Role of the J-domain in the cooperation of Hsp40 with Hsp70. Proc Natl Acad Sci U S A 95(11):6108–6113

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Harrison CJ, Hayer-Hartl M, Di Liberto M, Hartl F, Kuriyan J (1997) Crystal structure of the nucleotide exchange factor GrpE bound to the ATPase domain of the molecular chaperone DnaK. Science 276(5311):431–435

    Article  CAS  PubMed  Google Scholar 

  • Hartl FU, Hayer-Hartl M (2009) Converging concepts of protein folding in vitro and in vivo. Nat Struct Mol Biol 16(6):574–581

    Article  CAS  PubMed  Google Scholar 

  • Hendrickson WA, Liu Q (2008) Exchange we can believe in. Structure 16(8):1153–1155

    Article  CAS  PubMed  Google Scholar 

  • Jiang J, Maes EG, Taylor AB, Wang L, Hinck AP, Lafer EM, Sousa R (2007) Structural basis of J cochaperone binding and regulation of Hsp70. Mol Cell 28(3):422–433

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Kampinga HH, Craig EA (2010) The HSP70 chaperone machinery: J proteins as drivers of functional specificity. Nat Rev Mol Cell Biol 11(8):579–592

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Kim JH, Alderson TR, Frederick RO, Markley JL (2014) Nucleotide-dependent interactions within a specialized Hsp70/Hsp40 complex involved in Fe-S cluster biogenesis. J Am Chem Soc 136(33):11586–11589

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Kityk R, Kopp J, Sinning I, Mayer MP (2012) Structure and dynamics of the ATP-bound open conformation of Hsp70 chaperones. Mol Cell 48(6):863–874

    Article  CAS  PubMed  Google Scholar 

  • Kumar DP, Vorvis C, Sarbeng EB, Cabra Ledesma VC, Willis JE, Liu Q (2011) The four hydrophobic residues on the Hsp70 inter-domain linker have two distinct roles. J Mol Biol 411(5):1099–1113

    Article  CAS  PubMed  Google Scholar 

  • Landry SJ, Jordan R, McMacken R, Gierasch LM (1992) Different conformations for the same polypeptide bound to chaperones DnaK and GroEL. Nature 355(6359):455–457

    Article  CAS  PubMed  Google Scholar 

  • Laufen T, Mayer MP, Beisel C, Klostermeier D, Mogk A, Reinstein J, Bukau B (1999) Mechanism of regulation of hsp70 chaperones by DnaJ cochaperones. Proc Natl Acad Sci U S A 96(10):5452–5457

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Liberek K, Marszalek J, Ang D, Georgopoulos C, Zylicz M (1991) Escherichia coli DnaJ and GrpE heat shock proteins jointly stimulate ATPase activity of DnaK. Proc Natl Acad Sci U S A 88(7):2874–2878

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Liu Q, D’Silva P, Walter W, Marszalek J, Craig EA (2003) Regulated cycling of mitochondrial Hsp70 at the protein import channel. Science 300(5616):139–141

    Article  CAS  PubMed  Google Scholar 

  • Liu Q, Hendrickson WA (2007) Insights into Hsp70 chaperone activity from a crystal structure of the yeast Hsp110 Sse1. Cell 131(1):106–120

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Liu Q, Krzewska J, Liberek K, Craig EA (2001) Mitochondrial Hsp70 Ssc1: role in protein folding. J Biol Chem 276(9):6112–6118

    Article  CAS  PubMed  Google Scholar 

  • Mapa K, Sikor M, Kudryavtsev V, Waegemann K, Kalinin S, Seidel CA, Neupert W, Lamb DC, Mokranjac D (2010) The conformational dynamics of the mitochondrial Hsp70 chaperone. Mol Cell 38(1):89–100

    Article  CAS  PubMed  Google Scholar 

  • Mayer MP, Bukau B (2005) Hsp70 chaperones: cellular functions and molecular mechanism. Cell Mol Life Sci 62(6):670–684

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Mayer MP, Laufen T, Paal K, McCarty JS, Bukau B (1999) Investigation of the interaction between DnaK and DnaJ by surface plasmon resonance spectroscopy. J Mol Biol 289(4):1131–1144

    Article  CAS  PubMed  Google Scholar 

  • Montgomery DL, Morimoto RI, Gierasch LM (1999) Mutations in the substrate binding domain of the Escherichia coli 70 kDa molecular chaperone, DnaK, which alter substrate affinity or interdomain coupling. J Mol Biol 286(3):915–932

    Article  CAS  PubMed  Google Scholar 

  • Morgner N, Schmidt C, Beilsten-Edmands V, Ebong IO, Patel NA, Clerico EM, Kirschke E, Daturpalli S, Jackson SE, Agard D, Robinson CV (2015) Hsp70 forms antiparallel dimers stabilized by post-translational modifications to position clients for transfer to Hsp90. Cell Rep 11(5):759–769

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Mossessova E, Lima CD (2000) Ulp1-SUMO crystal structure and genetic analysis reveal conserved interactions and a regulatory element essential for cell growth in yeast. Mol Cell 5(5):865–876

    Article  CAS  PubMed  Google Scholar 

  • Muchowski PJ, Wacker JL (2005) Modulation of neurodegeneration by molecular chaperones. Nat Rev Neurosci 6(1):11–22

    Article  CAS  PubMed  Google Scholar 

  • Murphy ME (2013) The HSP70 family and cancer. Carcinogenesis 34(6):1181–1188

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Osipiuk J, Georgopoulos C, Zylicz M (1993) Initiation of lambda DNA replication. The Escherichia coli small heat shock proteins, DnaJ and GrpE, increase DnaK's affinity for the lambda P protein. J Biol Chem 268(7):4821–4827

    CAS  PubMed  Google Scholar 

  • Palleros DR, Reid KL, Shi L, Welch WJ, Fink AL (1993) ATP-induced protein-Hsp70 complex dissociation requires K+ but not ATP hydrolysis. Nature 365(6447):664–666

    Article  CAS  PubMed  Google Scholar 

  • Pellecchia M, Montgomery DL, Stevens SY, Vander Kooi CW, Feng HP, Gierasch LM, Zuiderweg ER (2000) Structural insights into substrate binding by the molecular chaperone DnaK. Nat Struct Biol 7(4):298–303

    Article  CAS  PubMed  Google Scholar 

  • Preissler S, Chambers JE, Crespillo-Casado A, Avezov E, Miranda E, Perez J, Hendershot LM, Harding HP, Ron D (2015) Physiological modulation of BiP activity by trans-protomer engagement of the interdomain linker. Elife 4:e08961

    PubMed  PubMed Central  Google Scholar 

  • Qi R, Sarbeng EB, Liu Q, Le KQ, Xu X, Xu H, Yang J, Wong JL, Vorvis C, Hendrickson WA, Zhou L, Liu Q (2013) Allosteric opening of the polypeptide-binding site when an Hsp70 binds ATP. Nat Struct Mol Biol 20(7):900–907

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Rudiger S, Germeroth L, Schneider-Mergener J, Bukau B (1997) Substrate specificity of the DnaK chaperone determined by screening cellulose-bound peptide libraries. EMBO J 16(7):1501–1507

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Sarbeng EB, Liu Q, Tian X, Yang J, Li H, Wong JL, Zhou L, Liu Q (2015) A functional DnaK dimer is essential for the efficient interaction with Hsp40 heat shock protein. J Biol Chem 290(14):8849–8862

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Schmid D, Baici A, Gehring H, Christen P (1994) Kinetics of molecular chaperone action. Science 263(5149):971–973

    Article  CAS  PubMed  Google Scholar 

  • Schonfeld HJ, Schmidt D, Schroder H, Bukau B (1995) The DnaK chaperone system of Escherichia coli: quaternary structures and interactions of the DnaK and GrpE components. J Biol Chem 270(5):2183–2189

    Article  CAS  PubMed  Google Scholar 

  • Schroder H, Langer T, Hartl FU, Bukau B (1993) DnaK, DnaJ and GrpE form a cellular chaperone machinery capable of repairing heat-induced protein damage. EMBO J 12(11):4137–4144

    CAS  PubMed  PubMed Central  Google Scholar 

  • Sriram M, Osipiuk J, Freeman B, Morimoto R, Joachimiak A (1997) Human Hsp70 molecular chaperone binds two calcium ions within the ATPase domain. Structure 5(3):403–414

    Article  CAS  PubMed  Google Scholar 

  • Suh WC, Burkholder WF, Lu CZ, Zhao X, Gottesman ME, Gross CA (1998) Interaction of the Hsp70 molecular chaperone, DnaK, with its cochaperone DnaJ. Proc Natl Acad Sci U S A 95(26):15223–15228

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Suh WC, Lu CZ, Gross CA (1999) Structural features required for the interaction of the Hsp70 molecular chaperone DnaK with its cochaperone DnaJ. J Biol Chem 274(43):30534–30539

    Article  CAS  PubMed  Google Scholar 

  • Swain JF, Dinler G, Sivendran R, Montgomery DL, Stotz M, Gierasch LM (2007) Hsp70 chaperone ligands control domain association via an allosteric mechanism mediated by the interdomain linker. Mol Cell 26(1):27–39

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Thompson AD, Bernard SM, Skiniotis G, Gestwicki JE (2012) Visualization and functional analysis of the oligomeric states of Escherichia coli heat shock protein 70 (Hsp70/DnaK). Cell Stress Chaperones 17(3):313–327

    Article  CAS  PubMed  Google Scholar 

  • Wawrzynow A, Zylicz M (1995) Divergent effects of ATP on the binding of the DnaK and DnaJ chaperones to each other, or to their various native and denatured protein substrates. J Biol Chem 270(33):19300–19306

    Article  CAS  PubMed  Google Scholar 

  • Wisen S, Bertelsen EB, Thompson AD, Patury S, Ung P, Chang L, Evans CG, Walter GM, Wipf P, Carlson HA, Brodsky JL, Zuiderweg ER, Gestwicki JE (2010) Binding of a small molecule at a protein-protein interface regulates the chaperone activity of hsp70-hsp40. ACS Chem Biol 5(6):611–622

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Wisniewska M, Karlberg T, Lehtio L, Johansson I, Kotenyova T, Moche M, Schuler H (2010) Crystal structures of the ATPase domains of four human Hsp70 isoforms: HSPA1L/Hsp70-hom, HSPA2/Hsp70-2, HSPA6/Hsp70B', and HSPA5/BiP/GRP78. PLoS One 5(1):e8625

    Article  PubMed  PubMed Central  Google Scholar 

  • Xu X, Sarbeng EB, Vorvis C, Kumar DP, Zhou L, Liu Q (2012) Unique peptide substrate binding properties of 110-kDa heat-shock protein (Hsp110) determine its distinct chaperone activity. J Biol Chem 287(8):5661–5672

    Article  CAS  PubMed  Google Scholar 

  • Yang J, Nune M, Zong Y, Zhou L, Liu Q (2015) Close and allosteric opening of the polypeptide-binding site in a human Hsp70 chaperone BiP. Structure 23(12):2191–2203

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Young JC (2010) Mechanisms of the Hsp70 chaperone system. Biochem Cell Biol 88(2):291–300

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Zhang P, Leu JI, Murphy ME, George DL, Marmorstein R (2014) Crystal structure of the stress-inducible human heat shock protein 70 substrate-binding domain in complex with peptide substrate. PLoS One 9(7):e103518

    Article  PubMed  PubMed Central  Google Scholar 

  • Zhu X, Zhao X, Burkholder WF, Gragerov A, Ogata CM, Gottesman ME, Hendrickson WA (1996) Structural analysis of substrate binding by the molecular chaperone DnaK. Science 272(5268):1606–1614

    Article  CAS  PubMed  Google Scholar 

  • Zhuravleva A, Clerico EM, Gierasch LM (2012) An interdomain energetic tug-of-war creates the allosterically active state in Hsp70 molecular chaperones. Cell 151(6):1296–1307

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Zhuravleva A, Gierasch LM (2015) Substrate-binding domain conformational dynamics mediate Hsp70 allostery. Proc Natl Acad Sci U S A 112(22):E2865–E2873

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

We thank Dr. Jiao Yang for discussion and Dr. Xueqing Liu for critically reading our manuscript. This work was supported by the National Institutes of Health (R01GM098592 to Qinglian Liu) and Blick Scholar Award from Virginia Commonwealth University (to Qinglian Liu). Qingdai Liu and Ying Yang were supported by the National Natural Science Foundation of China (no. 31571029). L.Z. is partially supported by RO1GM109193 from the National Institutes of Health.

Author information

Authors and Affiliations

  1. Key Laboratory of Food Nutrition and Safety, Tianjin University of Science and Technology, Ministry of Education, Tianjin, 300457, China

    Qingdai Liu, Ying Yang, Xueli Tian & Jiayue Su

  2. Department of Physiology and Biophysics, School of Medicine, Virginia Commonwealth University, Richmond, VA, 23298, USA

    Hongtao Li, Jiayue Su, Lei Zhou & Qinglian Liu

Authors
  1. Qingdai Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Hongtao Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Ying Yang
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Xueli Tian
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Jiayue Su
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Lei Zhou
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Qinglian Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to Qingdai Liu or Qinglian Liu.

Additional information

Qingdai Liu, Hongtao Li, Ying Yang, and Xueli Tian contributed equally to this work

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Liu, Q., Li, H., Yang, Y. et al. A disulfide-bonded DnaK dimer is maintained in an ATP-bound state. Cell Stress and Chaperones 22, 201–212 (2017). https://doi.org/10.1007/s12192-016-0752-y

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12192-016-0752-y

Keywords

Search

Navigation