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Human Stress-inducible Hsp70 Has a High Propensity to Form ATP-dependent Antiparallel Dimers That Are Differentially Regulated by Cochaperone Binding*[S]

  • Filip Trcka
    Affiliations
    Regional Centre for Applied Molecular Oncology, Masaryk Memorial Cancer Institute, Zluty kopec 7, 656 53 Brno, Czech Republic;
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  • Michal Durech
    Affiliations
    Regional Centre for Applied Molecular Oncology, Masaryk Memorial Cancer Institute, Zluty kopec 7, 656 53 Brno, Czech Republic;
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  • Pavla Vankova
    Affiliations
    BioCeV - Institute of Microbiology of the Czech Academy of Sciences, v.v.i., Prumyslova 595, 252 50 Vestec, Czech Republic;

    Department of Biochemistry, Faculty of Science, Charles University in Prague, Hlavova 8, 128 43 Prague, Czech Republic;
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  • Josef Chmelik
    Affiliations
    BioCeV - Institute of Microbiology of the Czech Academy of Sciences, v.v.i., Prumyslova 595, 252 50 Vestec, Czech Republic;

    Department of Biochemistry, Faculty of Science, Charles University in Prague, Hlavova 8, 128 43 Prague, Czech Republic;
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  • Veronika Martinkova
    Affiliations
    Regional Centre for Applied Molecular Oncology, Masaryk Memorial Cancer Institute, Zluty kopec 7, 656 53 Brno, Czech Republic;
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  • Jiri Hausner
    Affiliations
    BioCeV - Institute of Microbiology of the Czech Academy of Sciences, v.v.i., Prumyslova 595, 252 50 Vestec, Czech Republic;

    Department of Biochemistry, Faculty of Science, Charles University in Prague, Hlavova 8, 128 43 Prague, Czech Republic;
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  • Alan Kadek
    Footnotes
    Affiliations
    BioCeV - Institute of Microbiology of the Czech Academy of Sciences, v.v.i., Prumyslova 595, 252 50 Vestec, Czech Republic;

    Department of Biochemistry, Faculty of Science, Charles University in Prague, Hlavova 8, 128 43 Prague, Czech Republic;
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  • Julien Marcoux
    Affiliations
    Institut de Pharmacologie et de Biologie Structurale, Université de Toulouse, CNRS, UPS, Toulouse, France;
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  • Tomas Klumpler
    Affiliations
    CEITEC–Central European Institute of Technology, Masaryk University, 625 00 Brno, Czech Republic
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  • Borivoj Vojtesek
    Affiliations
    Regional Centre for Applied Molecular Oncology, Masaryk Memorial Cancer Institute, Zluty kopec 7, 656 53 Brno, Czech Republic;
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  • Petr Muller
    Correspondence
    To whom correspondence may be addressed..
    Affiliations
    Regional Centre for Applied Molecular Oncology, Masaryk Memorial Cancer Institute, Zluty kopec 7, 656 53 Brno, Czech Republic;
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  • Petr Man
    Correspondence
    To whom correspondence may be addressed.
    Affiliations
    BioCeV - Institute of Microbiology of the Czech Academy of Sciences, v.v.i., Prumyslova 595, 252 50 Vestec, Czech Republic;

    Department of Biochemistry, Faculty of Science, Charles University in Prague, Hlavova 8, 128 43 Prague, Czech Republic;
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  • Author Footnotes
    * This work was mainly supported by Czech Science Foundation (16-20860S), additional support was provided by the Ministry of Education, Youth and Sports of the Czech Republic (MEYS CR, LO1413, LQ1604 and LO1509), by the Ministry of Health of the Czech Republic - conceptual development of research organization (MMCI, 00209805) and EU CZ.1.05/1.1.00/02.0109 and OPPK CZ.2.16/3.1.00/24023. CIISB research infrastructure project LM2015043 funded by MEYS CR is gratefully acknowledged for the financial support of the measurements at the CF X-ray Diffraction and Bio-SAXS, CEITEC and at the Center of Molecular Structure, BioCeV. Part of the work was carried out with the support of Core Facility Biomolecular Interactions and Crystallization of CEITEC - Central European Institute of Technology under CEITEC - open access project LM2011020 funded by MEYS CR under the activity “Projects of major infrastructures for research, development and innovations”.
    [S] This article contains supplemental Figures and Tables.
    ‡‡ Current address: Heinrich Pette Institute – Leibniz Institute for Experimental Virology, Martinistraβe 52, 20251 Hamburg, Germany.
Open AccessPublished:November 20, 2018DOI:https://doi.org/10.1074/mcp.RA118.001044
      Eukaryotic protein homeostasis (proteostasis) is largely dependent on the action of highly conserved Hsp70 molecular chaperones. Recent evidence indicates that, apart from conserved molecular allostery, Hsp70 proteins have retained and adapted the ability to assemble as functionally relevant ATP-bound dimers throughout evolution. Here, we have compared the ATP-dependent dimerization of DnaK, human stress-inducible Hsp70, Hsc70 and BiP Hsp70 proteins, showing that their dimerization propensities differ, with stress-inducible Hsp70 being predominantly dimeric in the presence of ATP. Structural analyses using hydrogen/deuterium exchange mass spectrometry, native electrospray ionization mass spectrometry and small-angle X-ray scattering revealed that stress-inducible Hsp70 assembles in solution as an antiparallel dimer with the intermolecular interface closely resembling the ATP-bound dimer interfaces captured in DnaK and BiP crystal structures. ATP-dependent dimerization of stress-inducible Hsp70 is necessary for its efficient interaction with Hsp40, as shown by experiments with dimerization-deficient mutants. Moreover, dimerization of ATP-bound Hsp70 is required for its participation in high molecular weight protein complexes detected ex vivo, supporting its functional role in vivo. As human cytosolic Hsp70 can interact with tetratricopeptide repeat (TPR) domain containing cochaperones, we tested the interaction of Hsp70 ATP-dependent dimers with Chip and Tomm34 cochaperones. Although Chip associates with intact Hsp70 dimers to form a larger complex, binding of Tomm34 disrupts the Hsp70 dimer and this event plays an important role in Hsp70 activity regulation. In summary, this study provides structural evidence of robust ATP-dependent antiparallel dimerization of human inducible Hsp70 protein and suggests a novel role of TPR domain cochaperones in multichaperone complexes involving Hsp70 ATP-bound dimers.

      Graphical Abstract

      Protein homeostasis, including de novo protein synthesis surveillance, preprotein transport, misfolded protein degradation and aggregate dissolving, relies on the coordinated actions of the abundant Hsp70 and Hsp90 molecular chaperones (
      • Calamini B.
      • Morimoto R.I.
      Protein homeostasis as a therapeutic target for diseases of protein conformation.
      ,
      • Young J.C.
      • Agashe V.R.
      • Siegers K.
      • Hartl F.U.
      Pathways of chaperone-mediated protein folding in the cytosol.
      ). Hsp70 proteins exhibit remarkable sequence and structural conservation across all kingdoms of life, suggesting conserved molecular mechanics (
      • Young J.C.
      Mechanisms of the Hsp70 chaperone system.
      ,
      • Boorstein W.R.
      • Ziegelhoffer T.
      • Craig E.A.
      Molecular evolution of the HSP70 multigene family.
      ). In eukaryotes, Hsp70s have diversified into organelle-specific species including cytosolic Hsc70 and Hsp70 isoforms, and the endoplasmic reticulum (ER)
      The abbreviations used are:
      ER
      endoplasmic reticulum
      ADP
      adenosine diphosphate
      ATP
      adenosine triphosphate
      Bag-1
      Bcl2-associated athanogene 1
      Chip
      carboxyl terminus of Hsp70-interacting protein
      ESI-MS
      electrospray ionization mass spectrometry
      GST
      glutathione S-transferase
      HDX
      hydrogen/deuterium exchange
      Hsc
      heat shock cognate
      Hsp
      heat shock protein
      NBD
      nucleotide-binding domain
      NEF
      nucleotide exchange factor
      PDB
      protein data bank
      SAXS
      small-angle X-ray scattering
      SBD
      substrate-binding domain
      SBP
      streptavidin-binding peptide
      SEC
      size exclusion chromatography
      SPR
      surface plasmon resonance
      TEV
      tobacco etch virus
      Tomm34
      translocase of outer mitochondrial membrane 34
      TPR
      tetratricopeptide repeat
      WT
      wild type.
      1The abbreviations used are:ER
      endoplasmic reticulum
      ADP
      adenosine diphosphate
      ATP
      adenosine triphosphate
      Bag-1
      Bcl2-associated athanogene 1
      Chip
      carboxyl terminus of Hsp70-interacting protein
      ESI-MS
      electrospray ionization mass spectrometry
      GST
      glutathione S-transferase
      HDX
      hydrogen/deuterium exchange
      Hsc
      heat shock cognate
      Hsp
      heat shock protein
      NBD
      nucleotide-binding domain
      NEF
      nucleotide exchange factor
      PDB
      protein data bank
      SAXS
      small-angle X-ray scattering
      SBD
      substrate-binding domain
      SBP
      streptavidin-binding peptide
      SEC
      size exclusion chromatography
      SPR
      surface plasmon resonance
      TEV
      tobacco etch virus
      Tomm34
      translocase of outer mitochondrial membrane 34
      TPR
      tetratricopeptide repeat
      WT
      wild type.
      isoform, BiP (
      • Radons J.
      The human HSP70 family of chaperones: where do we stand?.
      ).
      Hsp70 proteins contain two independent domains: an N-terminal nucleotide-binding domain (NBD) and a C-terminal substrate-binding domain (SBD). The SBDβ subdomain forms a substrate-binding pocket with preference for hydrophobic polypeptide sequences (
      • Yang J.
      • Zong Y.
      • Su J.
      • Li H.
      • Zhu H.
      • Columbus L.
      • Zhou L.
      • Liu Q.
      Conformation transitions of the polypeptide-binding pocket support an active substrate release from Hsp70s.
      ,
      • Pellecchia M.
      • Montgomery D.L.
      • Stevens S.Y.
      • Vander Kooi C.W.
      • Feng H.P.
      • Gierasch L.M.
      • Zuiderweg E.R.
      Structural insights into substrate binding by the molecular chaperone DnaK.
      ), whereas SBDα forms an α-helical lid that covers SBDβ through ionic contacts and helps to stabilize substrate binding (
      • Fernandez-Saiz V.
      • Moro F.
      • Arizmendi J.M.
      • Acebron S.P.
      • Muga A.
      Ionic contacts at DnaK substrate binding domain involved in the allosteric regulation of lid dynamics.
      ,
      • Zhang P.
      • Leu J.I.
      • Murphy M.E.
      • George D.L.
      • Marmorstein R.
      Crystal structure of the stress-inducible human heat shock protein 70 substrate-binding domain in complex with peptide substrate.
      ). The NBD and SBD are connected by a highly conserved hydrophobic linker (
      • Swain J.F.
      • Dinler G.
      • Sivendran R.
      • Montgomery D.L.
      • Stotz M.
      • Gierasch L.M.
      Hsp70 chaperone ligands control domain association via an allosteric mechanism mediated by the interdomain linker.
      ). ATP coordination into the nucleotide-binding pocket of NBD leads to dramatic allosteric changes in Hsp70, characterized by NBD-SBDβ docking stabilizing the interdomain linker, accompanied by SBDα detachment from SBDβ and its docking onto the NBD (
      • Yang J.
      • Zong Y.
      • Su J.
      • Li H.
      • Zhu H.
      • Columbus L.
      • Zhou L.
      • Liu Q.
      Conformation transitions of the polypeptide-binding pocket support an active substrate release from Hsp70s.
      ,
      • Kityk R.
      • Kopp J.
      • Sinning I.
      • Mayer M.P.
      Structure and dynamics of the ATP-bound open conformation of Hsp70 chaperones.
      ,
      • Yang J.
      • Nune M.
      • Zong Y.
      • Zhou L.
      • Liu Q.
      Close and allosteric opening of the polypeptide-binding site in a human Hsp70 chaperone BiP.
      ,
      • Qi R.
      • Sarbeng E.B.
      • Liu Q.
      • Le K.Q.
      • Xu X.
      • Xu H.
      • Yang J.
      • Wong J.L.
      • Vorvis C.
      • Hendrickson W.A.
      • Zhou L.
      • Liu Q.
      Allosteric opening of the polypeptide-binding site when an Hsp70 binds ATP.
      ,
      • Liu Q.
      • Hendrickson W.A.
      Insights into Hsp70 chaperone activity from a crystal structure of the yeast Hsp110 Sse1.
      ). The SBDβ domain in the ATP-bound conformation of Hsp70 has unfavorable kinetics of substrate binding, which leads to substrate release (
      • Zhuravleva A.
      • Gierasch L.M.
      Substrate-binding domain conformational dynamics mediate Hsp70 allostery.
      ,
      • Buczynski G.
      • Slepenkov S.V.
      • Sehorn M.G.
      • Witt S.N.
      Characterization of a lidless form of the molecular chaperone DnaK: deletion of the lid increases peptide on- and off-rate constants.
      ). ATP hydrolysis triggers rearrangements in SBDβ reducing substrate on/off rates and therefore stabilizing substrate entrapment (
      • Mayer M.P.
      • Schroder H.
      • Rudiger S.
      • Paal K.
      • Laufen T.
      • Bukau B.
      Multistep mechanism of substrate binding determines chaperone activity of Hsp70.
      ,
      • Kityk R.
      • Kopp J.
      • Mayer M.P.
      Molecular Mechanism of J-Domain-Triggered ATP Hydrolysis by Hsp70 Chaperones.
      ,
      • Yu H.Y.
      • Ziegelhoffer T.
      • Craig E.A.
      Functionality of Class A and Class B J-protein cochaperones with Hsp70.
      ).
      Two classes of cochaperones modulate the ATPase cycle of Hsp70 by accelerating ATP hydrolysis and ADP/ATP nucleotide exchange: Hsp40 homologs and nucleotide exchange factors (NEFs) (
      • Yu H.Y.
      • Ziegelhoffer T.
      • Craig E.A.
      Functionality of Class A and Class B J-protein cochaperones with Hsp70.
      ,
      • Bracher A.
      • Verghese J.
      The nucleotide exchange factors of Hsp70 molecular chaperones.
      ). Hsp40 proteins contain a J-domain (named after the bacterial Hsp40 protein DnaJ) that interacts with the Hsp70 interdomain linker at the NBD-SBDβ interface, stimulating the NBD catalytic center (
      • Kityk R.
      • Kopp J.
      • Mayer M.P.
      Molecular Mechanism of J-Domain-Triggered ATP Hydrolysis by Hsp70 Chaperones.
      ). The J-domain also helps to communicate the hydrolysis-stimulating role of the substrate, which is brought to Hsp70 by Hsp40 substrate-binding activity (
      • Mayer M.P.
      • Schroder H.
      • Rudiger S.
      • Paal K.
      • Laufen T.
      • Bukau B.
      Multistep mechanism of substrate binding determines chaperone activity of Hsp70.
      ,
      • Kityk R.
      • Kopp J.
      • Mayer M.P.
      Molecular Mechanism of J-Domain-Triggered ATP Hydrolysis by Hsp70 Chaperones.
      ). A structurally differing group of NEFs assists ADP to ATP exchange in the NBD of Hsp70 by convergent mechanics through opening of the nucleotide binding cleft (
      • Bracher A.
      • Verghese J.
      The nucleotide exchange factors of Hsp70 molecular chaperones.
      ). Interesting members of the NEF group are Hsp110 proteins, represented by yeast Sse1, which exhibit significant structural homology to the ATP-bound state of Hsp70 and mediate nucleotide exchange through heterodimerization with Hsp70 (
      • Liu Q.
      • Hendrickson W.A.
      Insights into Hsp70 chaperone activity from a crystal structure of the yeast Hsp110 Sse1.
      ,
      • Yam A.Y.
      • Albanese V.
      • Lin H.T.
      • Frydman J.
      Hsp110 cooperates with different cytosolic HSP70 systems in a pathway for de novo folding.
      ).
      Beside the ATPase regulating cochaperones, eukaryotes have evolved a large group of cochaperones containing tetratricopeptide repeat (TPR) domains that mediate their interaction with the conserved C-terminal EEVD motif of eukaryotic Hsp70 and Hsp90 (
      • Scheufler C.
      • Brinker A.
      • Bourenkov G.
      • Pegoraro S.
      • Moroder L.
      • Bartunik H.
      • Hartl F.U.
      • Moarefi I.
      Structure of TPR domain-peptide complexes: critical elements in the assembly of the Hsp70-Hsp90 multichaperone machine.
      ). Apart from their TPR domains, these cochaperones bear additional domains with various functions diversifying molecular chaperones-mediated substrate processing (
      • Allan R.K.
      • Ratajczak T.
      Versatile TPR domains accommodate different modes of target protein recognition and function.
      ).
      Recently, dimerization and other oligomeric forms of Hsp70 family members in both conformational states, Apo/ADP- and ATP-bound, were described to play an important role in their chaperoning activities (
      • Aprile F.A.
      • Dhulesia A.
      • Stengel F.
      • Roodveldt C.
      • Benesch J.L.
      • Tortora P.
      • Robinson C.V.
      • Salvatella X.
      • Dobson C.M.
      • Cremades N.
      Hsp70 oligomerization is mediated by an interaction between the interdomain linker and the substrate-binding domain.
      ,
      • Sarbeng E.B.
      • Liu Q.
      • Tian X.
      • Yang J.
      • Li H.
      • Wong J.L.
      • Zhou L.
      • Liu Q.
      A functional DnaK dimer is essential for the efficient interaction with Hsp40 heat shock protein.
      ,
      • Liu Q.
      • Li H.
      • Yang Y.
      • Tian X.
      • Su J.
      • Zhou L.
      • Liu Q.
      A disulfide-bonded DnaK dimer is maintained in an ATP-bound state.
      ,
      • Morgner N.
      • Schmidt C.
      • Beilsten-Edmands V.
      • Ebong I.O.
      • Patel N.A.
      • Clerico E.M.
      • Kirschke E.
      • Daturpalli S.
      • Jackson S.E.
      • Agard D.
      • Robinson C.V.
      Hsp70 forms antiparallel dimers stabilized by post-translational modifications to position clients for transfer to Hsp90.
      ,
      • Preissler S.
      • Chambers J.E.
      • Crespillo-Casado A.
      • Avezov E.
      • Miranda E.
      • Perez J.
      • Hendershot L.M.
      • Harding H.P.
      • Ron D.
      Physiological modulation of BiP activity by trans-protomer engagement of the interdomain linker.
      ). Crystallographic studies revealed the presence of antiparallel ATP-bound dimers in the crystal structure of evolutionary distant Hsp70 orthologs DnaK (
      • Kityk R.
      • Kopp J.
      • Sinning I.
      • Mayer M.P.
      Structure and dynamics of the ATP-bound open conformation of Hsp70 chaperones.
      ,
      • Qi R.
      • Sarbeng E.B.
      • Liu Q.
      • Le K.Q.
      • Xu X.
      • Xu H.
      • Yang J.
      • Wong J.L.
      • Vorvis C.
      • Hendrickson W.A.
      • Zhou L.
      • Liu Q.
      Allosteric opening of the polypeptide-binding site when an Hsp70 binds ATP.
      ) and BiP (
      • Yang J.
      • Zong Y.
      • Su J.
      • Li H.
      • Zhu H.
      • Columbus L.
      • Zhou L.
      • Liu Q.
      Conformation transitions of the polypeptide-binding pocket support an active substrate release from Hsp70s.
      ,
      • Yang J.
      • Nune M.
      • Zong Y.
      • Zhou L.
      • Liu Q.
      Close and allosteric opening of the polypeptide-binding site in a human Hsp70 chaperone BiP.
      ). The ATP-dependent dimerization of DnaK is necessary for its cooperation with DnaJ, however the dimeric state is very low in solution (
      • Sarbeng E.B.
      • Liu Q.
      • Tian X.
      • Yang J.
      • Li H.
      • Wong J.L.
      • Zhou L.
      • Liu Q.
      A functional DnaK dimer is essential for the efficient interaction with Hsp40 heat shock protein.
      ). In contrast, our previous isothermal titration calorimetry (ITC) analysis suggested that human inducible Hsp70 isoform exists predominantly as a dimer in an ATP supplemented buffer (
      • Durech M.
      • Trcka F.
      • Man P.
      • Blackburn E.A.
      • Hernychova L.
      • Dvorakova P.
      • Coufalova D.
      • Kavan D.
      • Vojtesek B.
      • Muller P.
      Novel entropically driven conformation-specific interactions with Tomm34 protein modulate Hsp70 protein folding and ATPase activities.
      ).
      The aim of this work was to compare the levels of ATP-dependent dimerization in different Hsp70 proteins and to functionally and structurally characterize dimers of the human inducible Hsp70 isoform. Unlike DnaK and BiP, our data indicate that antiparallel dimerization of ATP-bound Hsp70 is highly pronounced in human Hsc70/Hsp70 cytosolic isoforms and is differentially regulated by interactions with Chip and Tomm34 TPR cochaperones.

      DISCUSSION

      Formation and disassembly of a small population of ATP-dependent DnaK dimers plays a critical role in DnaJ-regulated DnaK ATPase activity (
      • Sarbeng E.B.
      • Liu Q.
      • Tian X.
      • Yang J.
      • Li H.
      • Wong J.L.
      • Zhou L.
      • Liu Q.
      A functional DnaK dimer is essential for the efficient interaction with Hsp40 heat shock protein.
      ,
      • Liu Q.
      • Li H.
      • Yang Y.
      • Tian X.
      • Su J.
      • Zhou L.
      • Liu Q.
      A disulfide-bonded DnaK dimer is maintained in an ATP-bound state.
      ). Moreover, conservancy of residues involved in mediating homo-dimerization of ATP-bound DnaK in its eukaryotic homologs supports a functional role of the ATP-dependent dimer (
      • Malinverni D.
      • Marsili S.
      • Barducci A.
      • De Los Rios P.
      Large-scale conformational transitions and dimerization are encoded in the amino-acid sequences of Hsp70 chaperones.
      ). Here, we demonstrated that human Hsc70 and Hsp70 proteins form ATP-dependent dimers in solution with markedly higher efficiency compared with DnaK (Fig. 1A). The higher affinity of self-interactions between ATP-bound Hsp70 monomers versus Apo monomers is demonstrated by efficient ATP-bound dimer formation at low concentrations of Hsp70 (10 μm) compared with the more than 200 μm of Apo Hsp70 needed to assemble into dimers (Fig. 2A) (
      • Aprile F.A.
      • Dhulesia A.
      • Stengel F.
      • Roodveldt C.
      • Benesch J.L.
      • Tortora P.
      • Robinson C.V.
      • Salvatella X.
      • Dobson C.M.
      • Cremades N.
      Hsp70 oligomerization is mediated by an interaction between the interdomain linker and the substrate-binding domain.
      ,
      • Trcka F.
      • Durech M.
      • Man P.
      • Hernychova L.
      • Muller P.
      • Vojtesek B.
      The assembly and intermolecular properties of the Hsp70-Tomm34-Hsp90 molecular chaperone complex.
      ). Interestingly, Hsc70 was shown to follow ATP-dependent monomerization similarly to BiP in number of previous studies using SEC and SAXS (
      • Gao B.
      • Eisenberg E.
      • Greene L.
      Effect of constitutive 70-kDa heat shock protein polymerization on its interaction with protein substrate.
      ,
      • Benaroudj N.
      • Triniolles F.
      • Ladjimi M.M.
      Effect of nucleotides, peptides, and unfolded proteins on the self-association of the molecular chaperone HSC70.
      ,
      • Buxbaum E.
      • Woodman P.G.
      Binding of ATP and ATP analogues to the uncoating ATPase Hsc70 (70 kDa heat-shock cognate protein).
      ,
      • Wilbanks S.M.
      • Chen L.
      • Tsuruta H.
      • Hodgson K.O.
      • McKay D.B.
      Solution small-angle X-ray scattering study of the molecular chaperone Hsc70 and its subfragments.
      ). The discordance in the results might be caused by the fact that Hsc70 in those studies was purified from animal tissues and/or separated on Superose 12 SEC column. It has been demonstrated that posttranslational modifications and buffer ionic-strength influence Hsp70 ATP-dependent dimerization (
      • Morgner N.
      • Schmidt C.
      • Beilsten-Edmands V.
      • Ebong I.O.
      • Patel N.A.
      • Clerico E.M.
      • Kirschke E.
      • Daturpalli S.
      • Jackson S.E.
      • Agard D.
      • Robinson C.V.
      Hsp70 forms antiparallel dimers stabilized by post-translational modifications to position clients for transfer to Hsp90.
      ) and that Superose 12 ionically interacts with separated proteins complicating the interpretation (
      • Lee S.C.
      • Whitaker J.R.
      Are molecular weights of proteins determined by superose 12 column chromatography correct?.
      ). Alternatively, the presence of Hsp40 contamination in purified Hsc70 proteins would lead to rapid Hsc70 ATP-bound dimer dissociation after ATP addition (Fig. 2C). Hsp40 was also reported to promote ATP-dependent dimerization/oligomerization of Hsp70/Hsc70 purified from animal tissues (
      • King C.
      • Eisenberg E.
      • Greene L.
      Polymerization of 70-kDa heat shock protein by yeast DnaJ in ATP.
      ) or insect cells (
      • Morgner N.
      • Schmidt C.
      • Beilsten-Edmands V.
      • Ebong I.O.
      • Patel N.A.
      • Clerico E.M.
      • Kirschke E.
      • Daturpalli S.
      • Jackson S.E.
      • Agard D.
      • Robinson C.V.
      Hsp70 forms antiparallel dimers stabilized by post-translational modifications to position clients for transfer to Hsp90.
      ), indicating that the role of Hsp70 posttranslational modification might be decisive for its oligomerization properties. In addition, an SAXS study describing the structures of recombinant bacterially purified Hsc70 in ADP- and ATP-bound conformations revealed ATP-induced compaction of Hsc70 reflecting NBD-SBDβ docking in monomers (
      • Wilbanks S.M.
      • Chen L.
      • Tsuruta H.
      • Hodgson K.O.
      • McKay D.B.
      Solution small-angle X-ray scattering study of the molecular chaperone Hsc70 and its subfragments.
      ). However, the authors later discovered that the expression construct they used contained a mutation causing E543K substitution (
      • Ha J.H.
      • Hellman U.
      • Johnson E.R.
      • Li L.
      • McKay D.B.
      • Sousa M.C.
      • Takeda S.
      • Wernstedt C.
      • Wilbanks S.M.
      Destabilization of peptide binding and interdomain communication by an E543K mutation in the bovine 70-kDa heat shock cognate protein, a molecular chaperone.
      ). As we now demonstrate, E543 is crucial for ATP-dependent Hsp70 dimerization.
      The structural analyses (SAXS, HDX, Fig. 4, Fig. 5, and supplemental Fig. S3) with dimerization-deficient mutants of human Hsp70 (N540A, E543A, N540A-E543A, Fig. 3) revealed that Hsp70 predominantly assembles in solution as an antiparallel dimer, closely resembling the crystal packing of its homologs (
      • Yang J.
      • Zong Y.
      • Su J.
      • Li H.
      • Zhu H.
      • Columbus L.
      • Zhou L.
      • Liu Q.
      Conformation transitions of the polypeptide-binding pocket support an active substrate release from Hsp70s.
      ,
      • Kityk R.
      • Kopp J.
      • Sinning I.
      • Mayer M.P.
      Structure and dynamics of the ATP-bound open conformation of Hsp70 chaperones.
      ,
      • Yang J.
      • Nune M.
      • Zong Y.
      • Zhou L.
      • Liu Q.
      Close and allosteric opening of the polypeptide-binding site in a human Hsp70 chaperone BiP.
      ,
      • Qi R.
      • Sarbeng E.B.
      • Liu Q.
      • Le K.Q.
      • Xu X.
      • Xu H.
      • Yang J.
      • Wong J.L.
      • Vorvis C.
      • Hendrickson W.A.
      • Zhou L.
      • Liu Q.
      Allosteric opening of the polypeptide-binding site when an Hsp70 binds ATP.
      ,
      • Schuermann J.P.
      • Jiang J.
      • Cuellar J.
      • Llorca O.
      • Wang L.
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      ). A recent study suggested the presence of antiparallel human Hsp70 dimers in multichaperone complexes (
      • Morgner N.
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      • Beilsten-Edmands V.
      • Ebong I.O.
      • Patel N.A.
      • Clerico E.M.
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      Hsp70 forms antiparallel dimers stabilized by post-translational modifications to position clients for transfer to Hsp90.
      ), however their subunit orientation in the ATP-bound state inferred from chemical cross-linking does not satisfy the topology of antiparallel Hsp70 ATP-dependent dimers captured in DnaK/BiP structures and described here (
      • Yang J.
      • Zong Y.
      • Su J.
      • Li H.
      • Zhu H.
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      Conformation transitions of the polypeptide-binding pocket support an active substrate release from Hsp70s.
      ,
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      Structure and dynamics of the ATP-bound open conformation of Hsp70 chaperones.
      ,
      • Yang J.
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      • Zong Y.
      • Zhou L.
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      Close and allosteric opening of the polypeptide-binding site in a human Hsp70 chaperone BiP.
      ,
      • Qi R.
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      • Xu X.
      • Xu H.
      • Yang J.
      • Wong J.L.
      • Vorvis C.
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      Allosteric opening of the polypeptide-binding site when an Hsp70 binds ATP.
      ). This discrepancy could stem from the use of Hsp70 purified from eukaryotic expression system by Morgner et al. (
      • Morgner N.
      • Schmidt C.
      • Beilsten-Edmands V.
      • Ebong I.O.
      • Patel N.A.
      • Clerico E.M.
      • Kirschke E.
      • Daturpalli S.
      • Jackson S.E.
      • Agard D.
      • Robinson C.V.
      Hsp70 forms antiparallel dimers stabilized by post-translational modifications to position clients for transfer to Hsp90.
      ) (i.e. post-translationally modified) or be caused by the error-prone intra- and inter-molecular cross-links differentiation because of the homo-dimeric nature of Hsp70 (
      • Lima D.B.
      • Melchior J.T.
      • Morris J.
      • Barbosa V.C.
      • Chamot-Rooke J.
      • Fioramonte M.
      • Souza T.
      • Fischer J.S.G.
      • Gozzo F.C.
      • Carvalho P.C.
      • Davidson W.S.
      Characterization of homodimer interfaces with cross-linking mass spectrometry and isotopically labeled proteins.
      ). Although the residues mediating NBD-SBDα contacts in the antiparallel dimer are conserved, the NBD-NBD interfaces show lower conservation, as documented by inspection of crystal structures and our homology-based Hsp70 dimer model (supplemental Fig. S4). Therefore, NBD-SBDα contacts might serve as an elemental dimer interface that is accompanied by homolog-specific NBD-NBD interactions allowing for functional differentiation of Hsp70 dimeric species. Mechanistically, stable dimerization of ATP-bound Hsp70 may prevent Hsp70 monomers from premature association with Hsp110 and Bag NEFs as documented by evidence that Hsp110/BAG interfaces with Hsp70 NBD largely overlap with NBD-NBD contacts in the Hsp70 ATP-dependent dimer (
      • Sondermann H.
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      • Schneider C.
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      Structure of a Bag/Hsc70 complex: convergent functional evolution of Hsp70 nucleotide exchange factors.
      ,
      • Arakawa A.
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      • Kigawa T.
      • Hayashi F.
      • Shirouzu M.
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      The C-terminal BAG domain of BAG5 induces conformational changes of the Hsp70 nucleotide-binding domain for ADP-ATP exchange.
      ,
      • Williamson D.S.
      • Borgognoni J.
      • Clay A.
      • Daniels Z.
      • Dokurno P.
      • Drysdale M.J.
      • Foloppe N.
      • Francis G.L.
      • Graham C.J.
      • Howes R.
      • Macias A.T.
      • Murray J.B.
      • Parsons R.
      • Shaw T.
      • Surgenor A.E.
      • Terry L.
      • Wang Y.
      • Wood M.
      • Massey A.J.
      Novel adenosine-derived inhibitors of 70 kDa heat shock protein, discovered through structure-based design.
      ). Accordingly, although Bag1M protein increased the dissociation rate of ADP analog from Hsc70, its role in the dissociation of ATP analog was negligible (
      • Brehmer D.
      • Rudiger S.
      • Gassler C.S.
      • Klostermeier D.
      • Packschies L.
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      • Mayer M.P.
      • Bukau B.
      Tuning of chaperone activity of Hsp70 proteins by modulation of nucleotide exchange.
      ).
      Contrary to previous results that DnaK dimerization mutants N537A and D540A did not perturb substrate binding equilibria (
      • Sarbeng E.B.
      • Liu Q.
      • Tian X.
      • Yang J.
      • Li H.
      • Wong J.L.
      • Zhou L.
      • Liu Q.
      A functional DnaK dimer is essential for the efficient interaction with Hsp40 heat shock protein.
      ), N540, E543A and N540A-E543A substitutions in human Hsp70 severely compromised its substrate binding thermodynamics (Fig. 6A) because of a large destabilization of the Hsp70 SBDα region (Fig. 5 and supplemental Fig. S3). The faster kinetics of substrate binding for the dimerization mutants (Fig. 6B) resembles the behavior of lidless DnaK and the D540A-E548A DnaK mutant (
      • Fernandez-Saiz V.
      • Moro F.
      • Arizmendi J.M.
      • Acebron S.P.
      • Muga A.
      Ionic contacts at DnaK substrate binding domain involved in the allosteric regulation of lid dynamics.
      ,
      • Buczynski G.
      • Slepenkov S.V.
      • Sehorn M.G.
      • Witt S.N.
      Characterization of a lidless form of the molecular chaperone DnaK: deletion of the lid increases peptide on- and off-rate constants.
      ). Some DnaK/Hsp70 C-terminal truncation mutants exhibit intramolecular binding of the destabilized SBDα hydrophobic motifs into the substrate binding site of SBDβ (
      • Umehara K.
      • Hoshikawa M.
      • Tochio N.
      • Tate S.I.
      Substrate binding switches the conformation at the lynchpin site in the substrate-binding domain of human Hsp70 to enable allosteric interdomain communication.
      ,
      • Wang H.
      • Kurochkin A.V.
      • Pang Y.
      • Hu W.
      • Flynn G.C.
      • Zuiderweg E.R.
      NMR solution structure of the 21 kDa chaperone protein DnaK substrate binding domain: a preview of chaperone-protein interaction.
      ). This self-binding increases the intrinsic ATPase rate of these proteins (
      • Swain J.F.
      • Schulz E.G.
      • Gierasch L.M.
      Direct comparison of a stable isolated Hsp70 substrate-binding domain in the empty and substrate-bound states.
      ,
      • Sun L.
      • Edelmann F.T.
      • Kaiser C.J.
      • Papsdorf K.
      • Gaiser A.M.
      • Richter K.
      The lid domain of Caenorhabditis elegans Hsc70 influences ATP turnover, cofactor binding and protein folding activity.
      ,
      • Freeman B.C.
      • Myers M.P.
      • Schumacher R.
      • Morimoto R.I.
      Identification of a regulatory motif in Hsp70 that affects ATPase activity, substrate binding and interaction with HDJ-1.
      ). We do not expect the dimerization mutants to follow this mechanism because their basal ATPase activity is comparable to WT protein (Fig. 6D). Therefore, the elution profile of the predominantly monomeric N540A-E543A mutant (Fig. 3B) showing a dominant peak at MW 95 kDa in the presence of ATP is likely to mirror complex processes involving both impaired ATP-bound monomer/dimer equilibrium and destabilization of the SBDα subdomain (Fig. 5), rather than increased ATP hydrolysis by this mutant.
      Our data showed that the degree of dimerization defects in the analyzed mutants was mirrored by their decreased interaction with Hsp40 and refolding activity (Fig. 6CDEF). Dimerization deficient E543A and N540A-E543A mutants did not refold denatured luciferase even at a saturating concentration of Hsp40 (2 μm, Fig. 6C). Because N540A-E543A binds substrates very weakly (Fig. 6A and 6B), its inactivity in the refolding assay cannot be clearly distinguished from its dimerization and Hsp40 binding defects. However, although N540A and E543A have comparable defects in substrate binding activity (Fig. 6A), N540A mediates a significantly higher level of luciferase refolding (Fig. 6C). In addition, N540A mutation allows a significant level of Hsp70 dimerization and Hsp40 interaction (Fig. 3B, 3C, 3D, Fig. 6F). Therefore, we speculate that Hsp40-mediated substrate transfer to ATP-bound Hsp70 requires chaperone transient dimerization. Evidence exists suggesting that substrate binding to SBDβ needs to be followed by SBDα closing over SBDβ for effective Hsp40/substrate-stimulated ATPase activity of Hsp70 (
      • Kityk R.
      • Kopp J.
      • Sinning I.
      • Mayer M.P.
      Structure and dynamics of the ATP-bound open conformation of Hsp70 chaperones.
      ,
      • Liu Q.
      • Li H.
      • Yang Y.
      • Tian X.
      • Su J.
      • Zhou L.
      • Liu Q.
      A disulfide-bonded DnaK dimer is maintained in an ATP-bound state.
      ). Thus, transient stabilization of the SBDα subdomain of an Hsp70 protomer by its docking onto the NBD domain of the partner Hsp70 molecule might provide a time window for productive Hsp40-mediated substrate positioning into SBDβ before the SBDα subdomain closes over SBDβ. This hypothesis is consistent with the paradoxical observation that the DnaJ-DnaK interaction relies on both ATP-dependent DnaK dimerization (
      • Sarbeng E.B.
      • Liu Q.
      • Tian X.
      • Yang J.
      • Li H.
      • Wong J.L.
      • Zhou L.
      • Liu Q.
      A functional DnaK dimer is essential for the efficient interaction with Hsp40 heat shock protein.
      ) and dimer dissociation allowing free movement of the SBDα subdomain (
      • Liu Q.
      • Li H.
      • Yang Y.
      • Tian X.
      • Su J.
      • Zhou L.
      • Liu Q.
      A disulfide-bonded DnaK dimer is maintained in an ATP-bound state.
      ). Additionally, our SEC analysis of Hsp70 ATP-dependent dimers coincubated with Hsp40 (Fig. 2C) revealed complete dimer dissociation, however, the resulting monomers separated as both ATP-bound and ADP/Apo forms (apparent MW 80 and 95 kDa, respectively) indicating that Hsp40 dimer interaction with Hsp70 ATP-bound dimer does not activate the ATPase activity of both Hsp70 protomers. This observation suggests that Hsp702:Hsp402 interaction is asymmetrical.
      Hsp70 isolated from cells is commonly observed in larger protein complexes (
      • Faou P.
      • Hoogenraad N.J.
      Tom34: a cytosolic cochaperone of the Hsp90/Hsp70 protein complex involved in mitochondrial protein import.
      ,
      • Chavez J.D.
      • Schweppe D.K.
      • Eng J.K.
      • Bruce J.E.
      In Vivo Conformational Dynamics of Hsp90 and Its Interactors.
      ,
      • Klucken J.
      • Shin Y.
      • Masliah E.
      • Hyman B.T.
      • McLean P.J.
      Hsp70 Reduces alpha-Synuclein Aggregation and Toxicity.
      ). Our ex vivo experiments indicated that ATP-dependent Hsp70 dimerization is vital for Hsp70 participation in these high molecular weight complexes (Fig. 8A). Furthermore, antiparallel dimeric Hsp70 species were detected in in vitro reconstituted complexes involving Hsp90, Hop, Hsp40, and GR (Glucocorticoid receptor) (
      • Morgner N.
      • Schmidt C.
      • Beilsten-Edmands V.
      • Ebong I.O.
      • Patel N.A.
      • Clerico E.M.
      • Kirschke E.
      • Daturpalli S.
      • Jackson S.E.
      • Agard D.
      • Robinson C.V.
      Hsp70 forms antiparallel dimers stabilized by post-translational modifications to position clients for transfer to Hsp90.
      ), supporting the functional role of Hsp70 dimerization in multichaperone assemblies. The level of inducible Hsp70 is elevated under proteotoxic conditions and chronically increased in some pathologies, including cancer (
      • Sherman M.Y.
      • Gabai V.L.
      Hsp70 in cancer: back to the future.
      ,
      • Yaglom J.A.
      • Wang Y.
      • Li A.
      • Li Z.
      • Monti S.
      • Alexandrov I.
      • Lu X.
      • Sherman M.Y.
      Cancer cell responses to Hsp70 inhibitor JG-98: Comparison with Hsp90 inhibitors and finding synergistic drug combinations.
      ,
      • Kundrat L.
      • Regan L.
      Balance between folding and degradation for Hsp90-dependent client proteins: a key role for CHIP.
      ,
      • Kawazoe Y.
      • Nakai A.
      • Tanabe M.
      • Nagata K.
      Proteasome inhibition leads to the activation of all members of the heat-shock-factor family.
      ,
      • Woo J.A.
      • Liu T.
      • Zhao X.
      • Trotter C.
      • Yrigoin K.
      • Cazzaro S.
      • Narvaez E.
      • Khan H.
      • Witas R.
      • Bukhari A.
      • Makati K.
      • Wang X.
      • Dickey C.
      • Kang D.E.
      Enhanced tau pathology via RanBP9 and Hsp90/Hsc70 chaperone complexes.
      ,
      • Lackie R.E.
      • Maciejewski A.
      • Ostapchenko V.G.
      • Marques-Lopes J.
      • Choy W.Y.
      • Duennwald M.L.
      • Prado V.F.
      • Prado M.A.M.
      The Hsp70/Hsp90 Chaperone Machinery in Neurodegenerative Diseases.
      ). Given that intracellular concentrations of Hsp70 can reach up to tens of μm under stress conditions (
      • De Los Rios P.
      • Barducci A.
      Hsp70 chaperones are non-equilibrium machines that achieve ultra-affinity by energy consumption.
      ,
      • Nollen E.A.
      • Brunsting J.F.
      • Song J.
      • Kampinga H.H.
      • Morimoto R.I.
      Bag1 functions in vivo as a negative regulator of Hsp70 chaperone activity.
      ), the physiological importance of ATP-dependent Hsp70 dimers for overcoming proteotoxic stress becomes a likely hypothesis.
      Considering the recently discovered regulatory role of ATP-induced dimerization for Hsp70 chaperoning activity (Hsp40 binding) (
      • Sarbeng E.B.
      • Liu Q.
      • Tian X.
      • Yang J.
      • Li H.
      • Wong J.L.
      • Zhou L.
      • Liu Q.
      A functional DnaK dimer is essential for the efficient interaction with Hsp40 heat shock protein.
      ,
      • Liu Q.
      • Li H.
      • Yang Y.
      • Tian X.
      • Su J.
      • Zhou L.
      • Liu Q.
      A disulfide-bonded DnaK dimer is maintained in an ATP-bound state.
      ) and multichaperone complex formation (
      • Morgner N.
      • Schmidt C.
      • Beilsten-Edmands V.
      • Ebong I.O.
      • Patel N.A.
      • Clerico E.M.
      • Kirschke E.
      • Daturpalli S.
      • Jackson S.E.
      • Agard D.
      • Robinson C.V.
      Hsp70 forms antiparallel dimers stabilized by post-translational modifications to position clients for transfer to Hsp90.
      ), differential effects of TPR cochaperone binding to Hsp70 ATP-bound dimers (Fig. 7) are likely to be important for overall activity. Association of Hsp70 ATP-bound dimers with Chip may facilitate spatial coordination of Hsp40/Hsp70-mediated substrate processing and ubiquitination (
      • Zhang H.
      • Amick J.
      • Chakravarti R.
      • Santarriaga S.
      • Schlanger S.
      • McGlone C.
      • Dare M.
      • Nix J.C.
      • Scaglione K.M.
      • Stuehr D.J.
      • Misra S.
      • Page R.C.
      A bipartite interaction between Hsp70 and CHIP regulates ubiquitination of chaperoned client proteins.
      ,
      • Zhang H.T.
      • Zeng L.F.
      • He Q.Y.
      • Tao W.A.
      • Zha Z.G.
      • Hu C.D.
      The E3 ubiquitin ligase CHIP mediates ubiquitination and proteasomal degradation of PRMT5.
      ). Conversely, the inhibitory role of Tomm34 on Hsp70-mediated refolding activity (
      • Durech M.
      • Trcka F.
      • Man P.
      • Blackburn E.A.
      • Hernychova L.
      • Dvorakova P.
      • Coufalova D.
      • Kavan D.
      • Vojtesek B.
      • Muller P.
      Novel entropically driven conformation-specific interactions with Tomm34 protein modulate Hsp70 protein folding and ATPase activities.
      ) can be attributed to its ability to dissociate Hsp70 ATP-bound dimers (Fig. 7B and 7C) necessary for substrate capture during effective Hsp70-Hsp40 interaction (
      • Kityk R.
      • Kopp J.
      • Mayer M.P.
      Molecular Mechanism of J-Domain-Triggered ATP Hydrolysis by Hsp70 Chaperones.
      ,
      • Sarbeng E.B.
      • Liu Q.
      • Tian X.
      • Yang J.
      • Li H.
      • Wong J.L.
      • Zhou L.
      • Liu Q.
      A functional DnaK dimer is essential for the efficient interaction with Hsp40 heat shock protein.
      ,
      • Liu Q.
      • Li H.
      • Yang Y.
      • Tian X.
      • Su J.
      • Zhou L.
      • Liu Q.
      A disulfide-bonded DnaK dimer is maintained in an ATP-bound state.
      ,
      • Jin Y.
      • Awad W.
      • Petrova K.
      • Hendershot L.M.
      Regulated release of ERdj3 from unfolded proteins by BiP.
      ,
      • Petrova K.
      • Oyadomari S.
      • Hendershot L.M.
      • Ron D.
      Regulated association of misfolded endoplasmic reticulum lumenal proteins with P58/DNAJc3.
      ). We have previously suggested that the additional Tomm34-Hsp70 binding site is located between residues 533–543 in the SBDα subdomain (
      • Durech M.
      • Trcka F.
      • Man P.
      • Blackburn E.A.
      • Hernychova L.
      • Dvorakova P.
      • Coufalova D.
      • Kavan D.
      • Vojtesek B.
      • Muller P.
      Novel entropically driven conformation-specific interactions with Tomm34 protein modulate Hsp70 protein folding and ATPase activities.
      ). This region contains both residues (N540, E543) required for the formation of Hsp70 antiparallel dimers. It is yet to be determined whether Tomm34 occupies the 533–543 region directly or modulates its availability for Hsp70 homodimerization through allosteric changes in the SBDα subdomain. Nevertheless, ATP-dependent Hsp70 dimer dissociation by Tomm34 provides structural support for our previously suggested model of Tomm34-mediated pre-protein shuttling between Hsp70 and Hsp90 enabling its cytosolic transport in semifolded but aggregation-proof form (
      • Durech M.
      • Trcka F.
      • Man P.
      • Blackburn E.A.
      • Hernychova L.
      • Dvorakova P.
      • Coufalova D.
      • Kavan D.
      • Vojtesek B.
      • Muller P.
      Novel entropically driven conformation-specific interactions with Tomm34 protein modulate Hsp70 protein folding and ATPase activities.
      ). In addition, our results support the notion that TPR domain cochaperone binding to EEVD motif of Hsp70 does not follow a uniform scaffolding mechanism and suggest that the interaction is cochaperone specific (
      • Zhang H.
      • Amick J.
      • Chakravarti R.
      • Santarriaga S.
      • Schlanger S.
      • McGlone C.
      • Dare M.
      • Nix J.C.
      • Scaglione K.M.
      • Stuehr D.J.
      • Misra S.
      • Page R.C.
      A bipartite interaction between Hsp70 and CHIP regulates ubiquitination of chaperoned client proteins.
      ).
      The thermodynamics of Hsp70-Tomm34 interactions determined by our previous ITC analysis (
      • Durech M.
      • Trcka F.
      • Man P.
      • Blackburn E.A.
      • Hernychova L.
      • Dvorakova P.
      • Coufalova D.
      • Kavan D.
      • Vojtesek B.
      • Muller P.
      Novel entropically driven conformation-specific interactions with Tomm34 protein modulate Hsp70 protein folding and ATPase activities.
      ) is likely to reflect multiple equilibria transitions (e.g. Hsp70 ATP-bound monomer/dimer equilibrium), complicating data interpretation. Hsp70:Tomm34 complex stoichiometry was determined to be 2:1, whereas the current study shows that Tomm34 associates with ATP-bound Hsp70 monomer (Fig. 7B, 7C and 7D). A plausible explanation for this discrepancy would be that Hsp70 dimer dissociation by Tomm34 binding to one of the protomers renders the other protomer incapable of further association with Tomm34.
      In conclusion, ATP-dependent antiparallel Hsp70 dimerization is an evolutionary conserved mechanism for Hsp70 activity regulation that has been adapted by different Hsp70 homologs with various propensities. In contrast to bacterial DnaK and human BiP, human stress-induced Hsp70 is largely dimeric in the presence of ATP in vitro and ex vivo, which might reflect the specific role of stress-induced Hsp70 in the maintenance of cellular proteostasis under proteotoxic conditions. Moreover, Hsp70 ATP-bound dimers are used either as a scaffold for TPR domain cochaperone binding (Chip) or, conversely, cochaperone binding (Tomm34) leads to rapid disassembly, which may explain the inhibitory effect of Tomm34 on the Hsp70 chaperone system (
      • Durech M.
      • Trcka F.
      • Man P.
      • Blackburn E.A.
      • Hernychova L.
      • Dvorakova P.
      • Coufalova D.
      • Kavan D.
      • Vojtesek B.
      • Muller P.
      Novel entropically driven conformation-specific interactions with Tomm34 protein modulate Hsp70 protein folding and ATPase activities.
      ).

      DATA AVAILABILITY

      The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE (https://www.ebi.ac.uk/pride/archive/) partner repository with the dataset identifier PXD010069. Small angle scattering datasets, atomic model and fits have been deposited to the Small Angle Scattering Biological Data Bank (www.sasbdb.org) as entry SASDDN6.

      Acknowledgments

      We thank Dr. P. J. Coates for critical reading of the manuscript.

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