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Alternative modes of client binding enable functional plasticity of Hsp70

Abstract

The Hsp70 system is a central hub of chaperone activity in all domains of life. Hsp70 performs a plethora of tasks, including folding assistance, protection against aggregation, protein trafficking, and enzyme activity regulation1,2,3,4,5, and interacts with non-folded chains, as well as near-native, misfolded, and aggregated proteins6,7,8,9,10. Hsp70 is thought to achieve its many physiological roles by binding peptide segments that extend from these different protein conformers within a groove that can be covered by an ATP-driven helical lid11,12,13,14,15. However, it has been difficult to test directly how Hsp70 interacts with protein substrates in different stages of folding and how it affects their structure. Moreover, recent indications of diverse lid conformations in Hsp70–substrate complexes raise the possibility of additional interaction mechanisms15,16,17,18. Addressing these issues is technically challenging, given the conformational dynamics of both chaperone and client, the transient nature of their interaction, and the involvement of co-chaperones and the ATP hydrolysis cycle19. Here, using optical tweezers, we show that the bacterial Hsp70 homologue (DnaK) binds and stabilizes not only extended peptide segments, but also partially folded and near-native protein structures. The Hsp70 lid and groove act synergistically when stabilizing folded structures: stabilization is abolished when the lid is truncated and less efficient when the groove is mutated. The diversity of binding modes has important consequences: Hsp70 can both stabilize and destabilize folded structures, in a nucleotide-regulated manner; like Hsp90 and GroEL, Hsp70 can affect the late stages of protein folding; and Hsp70 can suppress aggregation by protecting partially folded structures as well as unfolded protein chains. Overall, these findings in the DnaK system indicate an extension of the Hsp70 canonical model that potentially affects a wide range of physiological roles of the Hsp70 system.

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Figure 1: DnaK chaperone system suppresses aggregation and promotes refolding.
Figure 2: DnaK binds and stabilizes folded structures.
Figure 3: DnaK lid is central to stabilizing folded structures and suppressing aggregation.
Figure 4: Model of Hsp70 chaperone action.

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Acknowledgements

Work in the group of S.T. is supported by the Foundation for Fundamental Research on Matter (FOM) and the Netherlands Organization for Scientific Research (NWO). Work in the laboratory of B.B. was supported by research grants from the Deutsche Forschungsgemeinschaft (SFB638 and FOR1805) to G.K. and B.B. The work in the laboratory of M.P.M. was funded by the Deutsche Forschungsgemeinschaft (MA 1278/4-1). We thank M. M. Naqvi for performing control experiments, M. Avellaneda for help with preparing protein structure illustrations, and T. Shimizu, E. Garnett and F. Huber for critical reading of the manuscript.

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Authors and Affiliations

Authors

Contributions

A.M., S.B., D.M., M.P.M., G.K., B.B. and S.T. conceived and designed the research; B.Z.B. and D.M. designed and purified the MBP protein constructs; A.M. and S.B. performed the optical tweezers experiments; B.Z.B., G.K., A.W. and R.K. purified the DnaK system chaperones and DnaK mutants; A.W. performed the bulk RepE54 assays; R.K. performed the bulk luciferase assays; A.M., S.B., A.W. and S.T. analysed the data; and A.M., S.B., D.M., M.P.M., G.K., B.B. and S.T. wrote the paper.

Corresponding author

Correspondence to Sander J. Tans.

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The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Domain architectures of DnaK, DnaJ and GrpE.

a, Structure of E. coli DnaK bound to peptide substrate and ADP nucleotide (PDB ID: 2KHO) in N-to-C-terminal colour gradient from red (N terminus) through dark grey to blue (C terminus)33. b, DnaJ domain structure containing J domain, glycine/phenylalanine-rich domain (G/F), cysteine-rich region/zinc-finger (CRR) and C-terminal domain; partial NMR structures of J domain (PDB ID: 1XBL) and crystal structure comprising cysteine-rich domain (CRR) and peptide-binding domain of the yeast homologue with co-crystalized peptide substrate highlighted in green spheres (PDB ID: 1NLT)34,35. c, GrpE structure (PDB ID: 1DKG)36.

Extended Data Figure 2 Canonical nucleotide-driven cycle of Hsp70–client interactions.

Hsp70 is known to interact with a large range of protein conformations from unfolded nascent chains to near-native proteins to aggregates and misfolded states. Non-native protein conformers are captured via short extended peptide segments by the substrate-binding groove of Hsp70. Hsp40 (DnaJ) then accelerates the ATP hydrolysis reaction together with the bound substrate. Hsp70–ADP locks the substrate under the closed helical lid of the substrate-binding domain in the ‘high-affinity’ state. The lid could also adopt the open conformation according to recent indications16,17,18,20. Nucleotide exchange factor (GrpE) subsequently accelerates the release of ADP and either spontaneous fluctuations of the lid or new ATP molecules facilitate the release of the substrate to regenerate Hsp70–ATP for another round of the cycle37.

Extended Data Figure 3 Protein structures.

a, DnaK in ADP state (PDB ID: 2KHO). Domain boundaries indicated in different colours: nucleotide-binding domain (purple, 1–383), hydrophobic linker (green, 384–396), β-sheet subdomain of substrate-binding domain with groove (orange, 397–502), α-helical lid subdomain of substrate binding domain (yellow, 503–602)38. b, DnaK in ATP bound open conformation (PDB ID: 4B9Q). c, Apo MBP (PDB ID: 2MV0). Proteins are displayed in the same scale to aid visualizing interactions between them.

Extended Data Figure 4 Predicted peptides binding to DnaK for MBP, luciferase and RepE54.

a, Peptide-library-trained predictions of DnaK-interacting peptide segments in the unfolded chain of MBP mapped in orange, using the algorithm introduced by Rüdiger et al.12 (PDB ID: 1ANF). N terminus marked green, C terminus marked blue. Surface representations of predicted peptides are restricted to their backbone atoms as their accessibility is central to canonical peptide binding of DnaK. b, Idem for MBP core structure. c, Idem for firefly luciferase (PDB ID: 1LCI) d, Idem for RepE54 (PDB ID: 1REP) including DNA ligand.

Extended Data Figure 5 Stabilization of folded structures by DnaK.

a, Schematic diagram of the setup. DnaK and ADP were present in the experimental buffer from the start of the experiment. b, In the first stretching curve (blue), only the C-terminal fragment of MBP was unfolded, and then force was immediately reduced to low force preventing further unfolding. After 3 min waiting at low force, subsequent stretching showed resistance to forced unfolding up to 50 pN (orange). Experiments performed in 100 nM DnaK with 1 mM ADP loading buffer. c, Force acting on MBP is plotted versus time. Pulls (single stretching–relaxation cycles) on the same MBP molecule are followed by increasing waiting periods at 0 pN, in the presence of 1 μM DnaK and 1 mM ADP loading buffer. d, Force–extension curves of MBP indicate increasing protection of partial folds against force (corresponding to panel c). Earlier pulls are in blue, later in red. e, Force on MBP versus time. Identical buffer conditions as panels c and d. f, Force–extension curves of MBP indicate the stabilization of compact state of MBP (corresponding to panel e). First pull is highlighted in blue, subsequent pulls in red. g, Force–extension curves of MBP in the presence of 100 nM DnaK and 1 mM ADP loading buffer. Blue, first stretching curves on different MBP molecules; orange and red, stretching curves after refolding and 3 min waiting at low force. h, i, MBP stretching and relaxation experiments in the presence of 1 μM DnaK with 100 μM ADP purified by high-performance liquid chromatography (HPLC) were present. First stretching curves are shown in blue, stretching curves denoted in shades of red were acquired after 3 min waiting at low force. j, MBP stretching and relaxation in the presence of 100 nM DnaK mutant T199A and 1 mM ATP, which traps DnaK in the ATP state. k, Corresponding refolding probability (n = 18). l, Force–extension curves of MBP in the presence of 1 μM lid-truncated DnaK and 1 mM ADP loading buffer, after refolding and 3 min waiting at low force (n = 13).

Extended Data Figure 6 DnaK mutant structures.

a, DnaK(V436F) (termed groove-mutated) structure model highlighting in red spheres the location of the point mutation hindering access to its groove, resulting in a 38-fold reduction in peptide-binding affinity24 (V436F substitution modelled onto PDB ID: 2KHO). b, DnaK(2–538) (termed lid-truncated) structure (lid deletion modelled using 2KHO). The part of the lid that is still present in DnaK(2–538) interacts with the nucleotide binding domain in the ATP-bound open conformation, which might be important for the mechanics of DnaK.

Extended Data Figure 7 4MBP refolding in the presence of ATP and wild-type or mutant DnaK.

a, Diagram of the experiment. bd, Stretching curves of 4MBP in the presence of ATP and wild-type DnaK (b), lid-truncated DnaK (c), and groove-mutated DnaK (d).

Extended Data Figure 8 MBP interaction with co-chaperone DnaJ and nucleotide exchange factor GrpE.

a, Diagram of the 4MBP experiments. b, Stretching curves of 4MBP with DnaJ. c, Diagram of the 1MBP experiments. d, 1MBP stretching in the presence of DnaJ. e, 1MBP stretching in the presence of GrpE.

Extended Data Figure 9 DnaK increases thermal stability of RepE54.

af, Thermal denaturation curves of RepE54 as measured by tryptophan fluorescence in the absence (a) or presence (b, c, f) of tryptophan-free DnaK(W102F) with ADP loading buffer (b), ADP (c), ATP (f) or lid-truncated (d) or groove-mutated (e) tryptophan-free DnaK with ADP loading buffer. Vertical lines mark the apparent melting points of RepE54 only (blue) and DnaK-stabilized RepE54 (red). Error bars indicate the standard error of the mean over three replicates.

Extended Data Figure 10 DnaK preserves enzyme activity.

Bulk luciferase protection functional assay monitoring luciferase activity at 37 °C in the absence or presence of nucleotides and chaperones as indicated. The active fraction of luciferase after 45 min from the start of temperature upshift from 0 °C to 37 °C is shown. The experiment is performed in triplicates; error bars indicate the standard error of the mean.

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Mashaghi, A., Bezrukavnikov, S., Minde, D. et al. Alternative modes of client binding enable functional plasticity of Hsp70. Nature 539, 448–451 (2016). https://doi.org/10.1038/nature20137

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