Heat shock-induced chaperoning by Hsp70 is enabled in-cell

Autoři: Drishti Guin aff001;  Hannah Gelman aff002;  Yuhan Wang aff003;  Martin Gruebele aff001
Působiště autorů: Department of Chemistry, University of Illinois Urbana-Champaign, Urbana, Illinois, United States of America aff001;  Department of Physics, University of Illinois Urbana-Champaign, Urbana, Illinois, United States of America aff002;  Center for Biophysics and Quantitative Biology, University of Illinois Urbana-Champaign, Urbana, Illinois, United States of America aff003
Vyšlo v časopise: PLoS ONE 14(9)
Kategorie: Research Article
doi: 10.1371/journal.pone.0222990


Recent work has shown that weak protein-protein interactions are susceptible to the cellular milieu. One case in point is the binding of heat shock proteins (Hsps) to substrate proteins in cells under stress. Upregulation of the Hsp70 chaperone machinery at elevated temperature was discovered in the 1960s, and more recent studies have shown that ATPase activity in one Hsp70 domain is essential for control of substrate binding by the other Hsp70 domain. Although there are several denaturant-based assays of Hsp70 activity, reports of ATP-dependent binding of Hsp70 to a globular protein substrate under heat shock are scarce. Here we show that binding of heat-inducible Hsp70 to phosphoglycerate kinase (PGK) is remarkably different in vitro compared to in-cell. We use fluorescent-labeled mHsp70 and ePGK, and begin by showing that mHsp70 passes the standard β-galactosidase assay, and that it does not self-aggregate until 50°C in presence of ATP. Yet during denaturant refolding or during in vitro heat shock, mHsp70 shows only ATP-independent non-specific sticking to ePGK, as evidenced by nearly identical results with an ATPase activity-deficient K71M mutant of Hsp70 as a control. Addition of Hsp40 (co-factor) or Ficoll (crowder) does not reduce non-specific sticking, but cell lysate does. Therefore, Hsp70 does not act as an ATP-dependent chaperone on its substrate PGK in vitro. In contrast, we observe only specific ATP-dependent binding of mHsp70 to ePGK in mammalian cells, when compared to the inactive Hsp70 K71M mutant. We hypothesize that enhanced in-cell activity is not due to an unknown co-factor, but simply to a favorable shift in binding equilibrium caused by the combination of crowding and osmolyte/macromolecular interactions present in the cell. One candidate mechanism for such a favorable shift in binding equilibrium is the proven ability of Hsp70 to bind near-native states of substrate proteins in vitro. We show evidence for early onset of binding in-cell. Our results suggest that Hsp70 binds PGK preemptively, prior to its full unfolding transition, thus stabilizing it against further unfolding. We propose a “preemptive holdase” mechanism for Hsp70-substrate binding. Given our result for PGK, more proteins than one might think based on in vitro assays may be chaperoned by Hsp70 in vivo. The cellular environment thus plays an important role in maintaining proper Hsp70 function.

Klíčová slova:

Adenosine triphosphatase – Cell binding – Heat shock response – Tryptophan – Fluorescence resonance energy transfer – Melting – Cell binding assay – Globular proteins


1. Zimmerman SB, Trach SO. Estimation of macromolecule concentrations and excluded volume effects for the cytoplasm of Escherichia coli. J Mol Biol. 1991;222: 599–620. doi: 10.1016/0022-2836(91)90499-v 1748995

2. Dhar A, Samiotakis A, Ebbinghaus S, Nienhaus L, Homouz D, Gruebele M, et al. Structure, function, and folding of phosphoglycerate kinase are strongly perturbed by macromolecular crowding. Proc Natl Acad Sci. 2010;107: 17586–17591. doi: 10.1073/pnas.1006760107 20921368

3. Monteith WB, Pielak GJ. Residue level quantification of protein stability in living cells. Proc Natl Acad Sci. 2014;111: 11335–11340. doi: 10.1073/pnas.1406845111 25049396

4. Guzman I, Gelman H, Tai J, Gruebele M. The extracellular protein VlsE is destabilized inside cells. J Mol Biol. 2014;426: 11–20. doi: 10.1016/j.jmb.2013.08.024 24013077

5. Mu X, Choi S, Lang L, Mowray D, Dokholyan N V., Danielsson J, et al. Physicochemical code for quinary protein interactions in Escherichia coli. Proc Natl Acad Sci. 2017;114: E4556–E4563. doi: 10.1073/pnas.1621227114 28536196

6. Palleros DR, Welch WJ, Fink AL. Interaction of hsp70 with unfolded proteins: effects of temperature and nucleotides on the kinetics of binding. Proc Natl Acad Sci. 1991;88: 5719–5723. doi: 10.1073/pnas.88.13.5719 1829527

7. Clerico EM, Tilitsky JM, Meng W, Gierasch LM. How hsp70 molecular machines interact with their substrates to mediate diverse physiological functions. J Mol Biol. 2015;427: 1575–88. doi: 10.1016/j.jmb.2015.02.004 25683596

8. Diamant S, Goloubinoff P. Temperature-Controlled Activity of DnaK−DnaJ−GrpE Chaperones: Protein-Folding Arrest and Recovery during and after Heat Shock Depends on the Substrate Protein and the GrpE Concentration. Biochemistry. 1998;37: 9688–9694. doi: 10.1021/bi980338u 9657681

9. Rosenzweig R, Sekhar A, Nagesh J, Kay LE. Promiscuous binding by Hsp70 results in conformational heterogeneity and fuzzy chaperone-substrate ensembles. Elife. eLife Sciences Publications, Ltd; 2017;6: e28030. doi: 10.7554/eLife.28030 28708484

10. Behnke J, Mann MJJ, Scruggs F-LL, Feige MJJ, Hendershot LMM. Members of the Hsp70 Family Recognize Distinct Types of Sequences to Execute ER Quality Control. Mol Cell. 2016;63: 739–752. doi: 10.1016/j.molcel.2016.07.012 27546788

11. Zhuravleva A, Gierasch LM. Substrate-binding domain conformational dynamics mediate Hsp70 allostery. Proc Natl Acad Sci. 2015;112: E2865–E2873. doi: 10.1073/pnas.1506692112 26038563

12. Carbonell P, Nussinov R, Del Sol A. Energetic determinants of protein binding specificity: Insights into protein interaction networks. Proteomics. 2009;9: 1744–1753. doi: 10.1002/pmic.200800425 19253304

13. Riback JA, Katanski CD, Kear-Scott JL, Pilipenko E V., Rojek AE, Sosnick TR, et al. Stress-Triggered Phase Separation Is an Adaptive. Evolutionarily Tuned Response, Cell. 2017;168: 1028–1040.e19. doi: 10.1016/j.cell.2017.02.027 28283059

14. Sukenik S, Ren P, Gruebele M. Weak protein–protein interactions in live cells are quantified by cell-volume modulation. Proc Natl Acad Sci. 2017;114: 6776–6781. doi: 10.1073/pnas.1700818114 28607089

15. McCarty JS, Buchberger A, Reinstein J, Bukau B. The Role of ATP in the Functional Cycle of the DnaK Chaperone System. J Mol Biol. 1995;249: 126–137. doi: 10.1006/jmbi.1995.0284 7776367

16. Hurley JH. The Sugar Kinase/Heat Shock Protein 70/Actin Superfamily: Implications of Conserved Structure for Mechanism. Annu Rev Biophys Biomol Struct. 1996;25: 137–162. doi: 10.1146/annurev.bb.25.060196.001033 8800467

17. Bork P, Sander C, Valencia A. An ATPase domain common to prokaryotic cell cycle proteins, sugar kinases, actin, and hsp70 heat shock proteins. Proc Natl Acad Sci U S A. National Academy of Sciences; 1992;89: 7290–4. doi: 10.1073/pnas.89.16.7290 1323828

18. Mayer MP, Schröder H, Rüdiger S, Paal K, Laufen T, Bukau B. Multistep mechanism of substrate binding determines chaperone activity of Hsp70. Nat Struct Biol. 2000;7: 586–593. doi: 10.1038/76819 10876246

19. Fourie AM, Sambrook JF, Gething MJH. Common and divergent peptide binding specificities of hsp70 molecular chaperones. J Biol Chem. 1994;269: 30470–30478. 7982963

20. Bösl B, Grimminger V, Walter S. Substrate binding to the molecular chaperone Hsp104 and its regulation by nucleotides. J Biol Chem. 2005;280: 38170–38176. doi: 10.1074/jbc.M506149200 16135516

21. Fernández-Higuero JA, Aguado A, Perales-Calvo J, Moro F, Muga A. Activation of the DnaK-ClpB Complex is Regulated by the Properties of the Bound Substrate. Sci Rep. 2018;8. doi: 10.1038/s41598-018-24140-5 29643454

22. Freeman BC, Michels A, Song J, Kampinga HH, Morimoto RI. Analysis of molecular chaperone activities using in vitro and in vivo approaches. Methods Mol Biol. New Jersey: Humana Press; 2000;99: 393–419. doi: 10.1385/1-59259-054-3:393

23. Palleros DR, Shi L, Reid KL, Fink a L. hsp70-protein complexes. Complex stability and conformation of bound substrate protein. J Biol Chem. 1994;269: 13107–14. Available: http://www.ncbi.nlm.nih.gov/pubmed/8175736 8175736

24. Mashaghi A, Bezrukavnikov S, Minde DP, Wentink AS, Kityk R, Zachmann-Brand B, et al. Alternative modes of client binding enable functional plasticity of Hsp70. Nature. Nature Research; 2016;539: 448–451. doi: 10.1038/nature20137 27783598

25. Sharma SK, De Los Rios P, Christen P, Lustig A, Goloubinoff P. The kinetic parameters and energy cost of the Hsp70 chaperone as a polypeptide unfoldase. Nat Chem Biol. Nature Research; 2010;6: 914–920. doi: 10.1038/nchembio.455 20953191

26. Schlecht R, Erbse AH, Bukau B, Mayer MP. Mechanics of Hsp70 chaperones enables differential interaction with client proteins. Nat Struct Mol Biol. 2011;18: 345–351. doi: 10.1038/nsmb.2006 21278757

27. Zhuravleva A, Clerico EM, Gierasch LM. An interdomain energetic tug-of-war creates the allosterically active state in Hsp70 molecular chaperones. Cell. 2012;151: 1296–1307. doi: 10.1016/j.cell.2012.11.002 23217711

28. Kityk R, Kopp J, Sinning I, Mayer MP. Structure and Dynamics of the ATP-Bound Open Conformation of Hsp70 Chaperones. Mol Cell. 2012;48: 863–874. doi: 10.1016/j.molcel.2012.09.023 23123194

29. Mogk a, Tomoyasu T, Goloubinoff P, Rüdiger S, Röder D, Langen H, et al. Identification of thermolabile Escherichia coli proteins: prevention and reversion of aggregation by DnaK and ClpB. EMBO J. 1999;18: 6934–6949. doi: 10.1093/emboj/18.24.6934 10601016

30. Kisley L, Serrano KA, Guin D, Kong X, Gruebele M, Leckband DE. Direct Imaging of Protein Stability and Folding Kinetics in Hydrogels. ACS Appl Mater Interfaces. 2017;9: 21606–21617. doi: 10.1021/acsami.7b01371 28553706

31. Ebbinghaus S, Dhar A, McDonald JD, Gruebele M. Protein folding stability and dynamics imaged in a living cell. Nat Methods. 2010;7: 319–323. doi: 10.1038/nmeth.1435 20190760

32. Schumacher RJ, Hurst R, Sullivan WP, McMahon NJ, Toft DO, Matts RL. ATP-dependent chaperoning activity of reticulocyte lysate. J Biol Chem. 1994;269: 9493–9499. 8144534

33. Greenfield NJ. Using circular dichroism spectra to estimate protein secondary structure. Nat Protoc. 2006;1: 2876–90. doi: 10.1038/nprot.2006.202 17406547

34. Wang S, Xie W, Rylander MN, Tucker PW, Aggarwal S, Diller KR. HSP70 kinetics study by continuous observation of HSP-GFP fusion protein expression on a perfusion heating stage. Biotechnol Bioeng. 2008;99: 146–154. doi: 10.1002/bit.21512 17546686

35. Dave K, Gelman H, Thu CTH, Guin D, Gruebele M. The Effect of Fluorescent Protein Tags on Phosphoglycerate Kinase Stability Is Nonadditive. J Phys Chem B. American Chemical Society; 2016;120: 2878–85. doi: 10.1021/acs.jpcb.5b11915 26923443

36. O’Brien MC, Flaherty KM, McKay DB. Lysine 71 of the chaperone protein Hsc70 Is essential for ATP hydrolysis. J Biol Chem. 1996;271: 15874–8. Available: http://www.ncbi.nlm.nih.gov/pubmed/8663302 doi: 10.1074/jbc.271.27.15874 8663302

37. Palleros DR, Reid KL, McCarty JS, Walker GC, Fink AL. DnaK, hsp73, and their molten globules. Two different ways heat shock proteins respond to heat. J Biol Chem. 1992;267: 5279–5285. 1544910

38. Dhar A, Girdhar K, Singh D, Gelman H, Ebbinghaus S, Gruebele M. Protein Stability and Folding Kinetics in the Nucleus and Endoplasmic Reticulum of Eucaryotic Cells. Biophys J. 2011;101: 421–430. doi: 10.1016/j.bpj.2011.05.071 21767495

39. Osváth S, Sabelko JJ, Gruebele M. Tuning the Heterogeneous Early Folding Dynamics of Phosphoglycerate Kinase. J Mol Biol. 2003;333: 187–199. doi: 10.1016/j.jmb.2003.08.011 14516752

40. Dhar A, Ebbinghaus S, Shen Z, Mishra T, Gruebele M. The Diffusion Coefficient for PGK Folding in Eukaryotic Cells. Biophys J. 2010;99: L69–L71. doi: 10.1016/j.bpj.2010.08.066 21044564

41. Mattoo RUH, Goloubinoff P. Molecular chaperones are nanomachines that catalytically unfold misfolded and alternatively folded proteins. Cell Mol Life Sci. Springer; 2014;71: 3311–25. doi: 10.1007/s00018-014-1627-y 24760129

42. Theodorakis NG, Morimoto RI. Posttranscriptional regulation of hsp70 expression in human cells: effects of heat shock, inhibition of protein synthesis, and adenovirus infection on translation and mRNA stability. Mol Cell Biol. 1987;7: 4357–4368. doi: 10.1128/mcb.7.12.4357 3437893

43. Flynn GC, Pohl J, Flocco MT, Rothman JE. Peptide-binding specificity of the molecular chaperone BiP. Nature. 1991;353: 726–730. doi: 10.1038/353726a0 1834945

44. Platkov M, Gruebele M. Periodic and stochastic thermal modulation of protein folding kinetics. J Chem Phys. American Institute of Physics; 2014;141: 035103. doi: 10.1063/1.4887360 25053342

45. Ervin J, Larios E, Osváth S, Schulten K, Gruebele M. What causes hyperfluorescence: folding intermediates or conformationally flexible native states? Biophys J. Cell Press; 2002;83: 473–83. doi: 10.1016/S0006-3495(02)75183-7 12080134

46. Inoue R, Biehl R, Rosenkranz T, Fitter J, Monkenbusch M, Radulescu A, et al. Large Domain Fluctuations on 50-ns Timescale Enable Catalytic Activity in Phosphoglycerate Kinase. Biophys J. 2010;99: 2309–2317. doi: 10.1016/j.bpj.2010.08.017 20923666

47. Sekhar A, Velyvis A, Zoltsman G, Rosenzweig R, Bouvignies G, Kay LE. Conserved conformational selection mechanism of Hsp70 chaperone-substrate interactions. Elife. eLife Sciences Publications Limited; 2018;7: e32764. doi: 10.7554/eLife.32764 29460778

48. Van Durme J, Maurer-Stroh S, Gallardo R, Wilkinson H, Rousseau F, Schymkowitz J. Accurate prediction of DnaK-peptide binding via homology modelling and experimental data. PLoS Comput Biol. 2009; doi: 10.1371/journal.pcbi.1000475 19696878

49. Monteith WB, Cohen RD, Smith AE, Guzman-Cisneros E, Pielak GJ. Quinary structure modulates protein stability in cells. Proc Natl Acad Sci U S A. National Academy of Sciences; 2015;112: 1739–42. doi: 10.1073/pnas.1417415112 25624496

50. Wirth AJ, Gruebele M. Quinary protein structure and the consequences of crowding in living cells: leaving the test-tube behind. BioEssays. 2013;35: 984–993. doi: 10.1002/bies.201300080 23943406

51. Chien P, Gierasch LM. Challenges and dreams: physics of weak interactions essential to life. Mol Biol Cell. American Society for Cell Biology; 2014;25: 3474–7. doi: 10.1091/mbc.E14-06-1035 25368424

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