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. 1999 Feb;8(2):343–354. doi: 10.1110/ps.8.2.343

Topology and dynamics of the 10 kDa C-terminal domain of DnaK in solution.

E B Bertelsen 1, H Zhou 1, D F Lowry 1, G C Flynn 1, F W Dahlquist 1
PMCID: PMC2144266  PMID: 10048327

Abstract

Hsp70 molecular chaperones contain three distinct structural domains, a 44 kDa N-terminal ATPase domain, a 17 kDa peptide-binding domain, and a 10 kDa C-terminal domain. The ATPase and peptide binding domains are conserved in sequence and are functionally well characterized. The function of the 10 kDa variable C-terminal domain is less well understood. We have characterized the secondary structure and dynamics of the C-terminal domain from the Escherichia coli Hsp70, DnaK, in solution by high-resolution NMR. The domain was shown to be comprised of a rigid structure consisting of four helices and a flexible C-terminal subdomain of approximately 33 amino acids. The mobility of the flexible region is maintained in the context of the full-length protein and does not appear to be modulated by the nucleotide state. The flexibility of this region appears to be a conserved feature of Hsp70 architecture and may have important functional implications. We also developed a method to analyze 15N nuclear spin relaxation data, which allows us to extract amide bond vector directions relative to a unique diffusion axis. The extracted angles and rotational correlation times indicate that the helices form an elongated, bundle-like structure in solution.

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Selected References

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  1. Bai Y., Milne J. S., Mayne L., Englander S. W. Primary structure effects on peptide group hydrogen exchange. Proteins. 1993 Sep;17(1):75–86. doi: 10.1002/prot.340170110. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Barbato G., Ikura M., Kay L. E., Pastor R. W., Bax A. Backbone dynamics of calmodulin studied by 15N relaxation using inverse detected two-dimensional NMR spectroscopy: the central helix is flexible. Biochemistry. 1992 Jun 16;31(23):5269–5278. doi: 10.1021/bi00138a005. [DOI] [PubMed] [Google Scholar]
  3. Billeter M., Neri D., Otting G., Qian Y. Q., Wüthrich K. Precise vicinal coupling constants 3JHN alpha in proteins from nonlinear fits of J-modulated [15N,1H]-COSY experiments. J Biomol NMR. 1992 May;2(3):257–274. doi: 10.1007/BF01875320. [DOI] [PubMed] [Google Scholar]
  4. Bolliger L., Deloche O., Glick B. S., Georgopoulos C., Jenö P., Kronidou N., Horst M., Morishima N., Schatz G. A mitochondrial homolog of bacterial GrpE interacts with mitochondrial hsp70 and is essential for viability. EMBO J. 1994 Apr 15;13(8):1998–2006. doi: 10.1002/j.1460-2075.1994.tb06469.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Boorstein W. R., Ziegelhoffer T., Craig E. A. Molecular evolution of the HSP70 multigene family. J Mol Evol. 1994 Jan;38(1):1–17. doi: 10.1007/BF00175490. [DOI] [PubMed] [Google Scholar]
  6. Brüschweiler R., Liao X., Wright P. E. Long-range motional restrictions in a multidomain zinc-finger protein from anisotropic tumbling. Science. 1995 May 12;268(5212):886–889. doi: 10.1126/science.7754375. [DOI] [PubMed] [Google Scholar]
  7. Buchberger A., Theyssen H., Schröder H., McCarty J. S., Virgallita G., Milkereit P., Reinstein J., Bukau B. Nucleotide-induced conformational changes in the ATPase and substrate binding domains of the DnaK chaperone provide evidence for interdomain communication. J Biol Chem. 1995 Jul 14;270(28):16903–16910. doi: 10.1074/jbc.270.28.16903. [DOI] [PubMed] [Google Scholar]
  8. Bukau B., Walker G. C. Mutations altering heat shock specific subunit of RNA polymerase suppress major cellular defects of E. coli mutants lacking the DnaK chaperone. EMBO J. 1990 Dec;9(12):4027–4036. doi: 10.1002/j.1460-2075.1990.tb07624.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Caplan A. J., Cyr D. M., Douglas M. G. Eukaryotic homologues of Escherichia coli dnaJ: a diverse protein family that functions with hsp70 stress proteins. Mol Biol Cell. 1993 Jun;4(6):555–563. doi: 10.1091/mbc.4.6.555. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Chappell T. G., Konforti B. B., Schmid S. L., Rothman J. E. The ATPase core of a clathrin uncoating protein. J Biol Chem. 1987 Jan 15;262(2):746–751. [PubMed] [Google Scholar]
  11. Clore G. M., Driscoll P. C., Wingfield P. T., Gronenborn A. M. Analysis of the backbone dynamics of interleukin-1 beta using two-dimensional inverse detected heteronuclear 15N-1H NMR spectroscopy. Biochemistry. 1990 Aug 14;29(32):7387–7401. doi: 10.1021/bi00484a006. [DOI] [PubMed] [Google Scholar]
  12. Clore G. M., Gronenborn A. M. New methods of structure refinement for macromolecular structure determination by NMR. Proc Natl Acad Sci U S A. 1998 May 26;95(11):5891–5898. doi: 10.1073/pnas.95.11.5891. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Farrow N. A., Muhandiram R., Singer A. U., Pascal S. M., Kay C. M., Gish G., Shoelson S. E., Pawson T., Forman-Kay J. D., Kay L. E. Backbone dynamics of a free and phosphopeptide-complexed Src homology 2 domain studied by 15N NMR relaxation. Biochemistry. 1994 May 17;33(19):5984–6003. doi: 10.1021/bi00185a040. [DOI] [PubMed] [Google Scholar]
  14. Farrow N. A., Zhang O., Forman-Kay J. D., Kay L. E. Characterization of the backbone dynamics of folded and denatured states of an SH3 domain. Biochemistry. 1997 Mar 4;36(9):2390–2402. doi: 10.1021/bi962548h. [DOI] [PubMed] [Google Scholar]
  15. Farrow N. A., Zhang O., Forman-Kay J. D., Kay L. E. Comparison of the backbone dynamics of a folded and an unfolded SH3 domain existing in equilibrium in aqueous buffer. Biochemistry. 1995 Jan 24;34(3):868–878. doi: 10.1021/bi00003a021. [DOI] [PubMed] [Google Scholar]
  16. Flaherty K. M., DeLuca-Flaherty C., McKay D. B. Three-dimensional structure of the ATPase fragment of a 70K heat-shock cognate protein. Nature. 1990 Aug 16;346(6285):623–628. doi: 10.1038/346623a0. [DOI] [PubMed] [Google Scholar]
  17. 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. EMBO J. 1995 May 15;14(10):2281–2292. doi: 10.1002/j.1460-2075.1995.tb07222.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Gething M. J., Sambrook J. Protein folding in the cell. Nature. 1992 Jan 2;355(6355):33–45. doi: 10.1038/355033a0. [DOI] [PubMed] [Google Scholar]
  19. Gettins P., Cunningham L. W. Identification of 1H resonances from the bait region of human alpha 2-macroglobulin and effects of proteases and methylamine. Biochemistry. 1986 Sep 9;25(18):5011–5017. doi: 10.1021/bi00366a007. [DOI] [PubMed] [Google Scholar]
  20. Gragerov A., Nudler E., Komissarova N., Gaitanaris G. A., Gottesman M. E., Nikiforov V. Cooperation of GroEL/GroES and DnaK/DnaJ heat shock proteins in preventing protein misfolding in Escherichia coli. Proc Natl Acad Sci U S A. 1992 Nov 1;89(21):10341–10344. doi: 10.1073/pnas.89.21.10341. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Ha J. H., McKay D. B. ATPase kinetics of recombinant bovine 70 kDa heat shock cognate protein and its amino-terminal ATPase domain. Biochemistry. 1994 Dec 6;33(48):14625–14635. doi: 10.1021/bi00252a031. [DOI] [PubMed] [Google Scholar]
  22. Harrison C. J., Hayer-Hartl M., Di Liberto M., Hartl F., Kuriyan J. Crystal structure of the nucleotide exchange factor GrpE bound to the ATPase domain of the molecular chaperone DnaK. Science. 1997 Apr 18;276(5311):431–435. doi: 10.1126/science.276.5311.431. [DOI] [PubMed] [Google Scholar]
  23. Hendrick J. P., Hartl F. U. The role of molecular chaperones in protein folding. FASEB J. 1995 Dec;9(15):1559–1569. doi: 10.1096/fasebj.9.15.8529835. [DOI] [PubMed] [Google Scholar]
  24. Höhfeld J., Minami Y., Hartl F. U. Hip, a novel cochaperone involved in the eukaryotic Hsc70/Hsp40 reaction cycle. Cell. 1995 Nov 17;83(4):589–598. doi: 10.1016/0092-8674(95)90099-3. [DOI] [PubMed] [Google Scholar]
  25. Karzai A. W., McMacken R. A bipartite signaling mechanism involved in DnaJ-mediated activation of the Escherichia coli DnaK protein. J Biol Chem. 1996 May 10;271(19):11236–11246. doi: 10.1074/jbc.271.19.11236. [DOI] [PubMed] [Google Scholar]
  26. Kassenbrock C. K., Kelly R. B. Interaction of heavy chain binding protein (BiP/GRP78) with adenine nucleotides. EMBO J. 1989 May;8(5):1461–1467. doi: 10.1002/j.1460-2075.1989.tb03529.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Kay L. E., Torchia D. A., Bax A. Backbone dynamics of proteins as studied by 15N inverse detected heteronuclear NMR spectroscopy: application to staphylococcal nuclease. Biochemistry. 1989 Nov 14;28(23):8972–8979. doi: 10.1021/bi00449a003. [DOI] [PubMed] [Google Scholar]
  28. Landry S. J., Taher A., Georgopoulos C., van der Vies S. M. Interplay of structure and disorder in cochaperonin mobile loops. Proc Natl Acad Sci U S A. 1996 Oct 15;93(21):11622–11627. doi: 10.1073/pnas.93.21.11622. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Landry S. J., Zeilstra-Ryalls J., Fayet O., Georgopoulos C., Gierasch L. M. Characterization of a functionally important mobile domain of GroES. Nature. 1993 Jul 15;364(6434):255–258. doi: 10.1038/364255a0. [DOI] [PubMed] [Google Scholar]
  30. Liberek K., Marszalek J., Ang D., Georgopoulos C., Zylicz M. Escherichia coli DnaJ and GrpE heat shock proteins jointly stimulate ATPase activity of DnaK. Proc Natl Acad Sci U S A. 1991 Apr 1;88(7):2874–2878. doi: 10.1073/pnas.88.7.2874. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. McCarty J. S., Buchberger A., Reinstein J., Bukau B. The role of ATP in the functional cycle of the DnaK chaperone system. J Mol Biol. 1995 May 26;249(1):126–137. doi: 10.1006/jmbi.1995.0284. [DOI] [PubMed] [Google Scholar]
  32. McEvoy M. M., de la Cruz A. F., Dahlquist F. W. Large modular proteins by NMR. Nat Struct Biol. 1997 Jan;4(1):9–9. doi: 10.1038/nsb0197-9. [DOI] [PubMed] [Google Scholar]
  33. Mensa-Wilmot K., Seaby R., Alfano C., Wold M. C., Gomes B., McMacken R. Reconstitution of a nine-protein system that initiates bacteriophage lambda DNA replication. J Biol Chem. 1989 Feb 15;264(5):2853–2861. [PubMed] [Google Scholar]
  34. Morshauser R. C., Wang H., Flynn G. C., Zuiderweg E. R. The peptide-binding domain of the chaperone protein Hsc70 has an unusual secondary structure topology. Biochemistry. 1995 May 16;34(19):6261–6266. doi: 10.1021/bi00019a001. [DOI] [PubMed] [Google Scholar]
  35. Muchmore D. C., McIntosh L. P., Russell C. B., Anderson D. E., Dahlquist F. W. Expression and nitrogen-15 labeling of proteins for proton and nitrogen-15 nuclear magnetic resonance. Methods Enzymol. 1989;177:44–73. doi: 10.1016/0076-6879(89)77005-1. [DOI] [PubMed] [Google Scholar]
  36. Palleros D. R., Reid K. L., Shi L., Fink A. L. DnaK ATPase activity revisited. FEBS Lett. 1993 Dec 20;336(1):124–128. doi: 10.1016/0014-5793(93)81624-9. [DOI] [PubMed] [Google Scholar]
  37. Pellecchia M., Szyperski T., Wall D., Georgopoulos C., Wüthrich K. NMR structure of the J-domain and the Gly/Phe-rich region of the Escherichia coli DnaJ chaperone. J Mol Biol. 1996 Jul 12;260(2):236–250. doi: 10.1006/jmbi.1996.0395. [DOI] [PubMed] [Google Scholar]
  38. Pontius B. W. Close encounters: why unstructured, polymeric domains can increase rates of specific macromolecular association. Trends Biochem Sci. 1993 May;18(5):181–186. doi: 10.1016/0968-0004(93)90111-y. [DOI] [PubMed] [Google Scholar]
  39. Russell R., Jordan R., McMacken R. Kinetic characterization of the ATPase cycle of the DnaK molecular chaperone. Biochemistry. 1998 Jan 13;37(2):596–607. doi: 10.1021/bi972025p. [DOI] [PubMed] [Google Scholar]
  40. Rüdiger S., Buchberger A., Bukau B. Interaction of Hsp70 chaperones with substrates. Nat Struct Biol. 1997 May;4(5):342–349. doi: 10.1038/nsb0597-342. [DOI] [PubMed] [Google Scholar]
  41. Schönfeld H. J., Schmidt D., Schröder H., Bukau B. The DnaK chaperone system of Escherichia coli: quaternary structures and interactions of the DnaK and GrpE components. J Biol Chem. 1995 Feb 3;270(5):2183–2189. doi: 10.1074/jbc.270.5.2183. [DOI] [PubMed] [Google Scholar]
  42. Silver P. A., Way J. C. Eukaryotic DnaJ homologs and the specificity of Hsp70 activity. Cell. 1993 Jul 16;74(1):5–6. doi: 10.1016/0092-8674(93)90287-z. [DOI] [PubMed] [Google Scholar]
  43. Stuart R. A., Cyr D. M., Craig E. A., Neupert W. Mitochondrial molecular chaperones: their role in protein translocation. Trends Biochem Sci. 1994 Feb;19(2):87–92. doi: 10.1016/0968-0004(94)90041-8. [DOI] [PubMed] [Google Scholar]
  44. Szyperski T., Pellecchia M., Wall D., Georgopoulos C., Wüthrich K. NMR structure determination of the Escherichia coli DnaJ molecular chaperone: secondary structure and backbone fold of the N-terminal region (residues 2-108) containing the highly conserved J domain. Proc Natl Acad Sci U S A. 1994 Nov 22;91(24):11343–11347. doi: 10.1073/pnas.91.24.11343. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Tjandra N., Bax A. Direct measurement of distances and angles in biomolecules by NMR in a dilute liquid crystalline medium. Science. 1997 Nov 7;278(5340):1111–1114. doi: 10.1126/science.278.5340.1111. [DOI] [PubMed] [Google Scholar]
  46. Tjandra N., Garrett D. S., Gronenborn A. M., Bax A., Clore G. M. Defining long range order in NMR structure determination from the dependence of heteronuclear relaxation times on rotational diffusion anisotropy. Nat Struct Biol. 1997 Jun;4(6):443–449. doi: 10.1038/nsb0697-443. [DOI] [PubMed] [Google Scholar]
  47. Tsai M. Y., Wang C. Uncoupling of peptide-stimulated ATPase and clathrin-uncoating activity in deletion mutant of hsc70. J Biol Chem. 1994 Feb 25;269(8):5958–5962. [PubMed] [Google Scholar]
  48. Wall D., Zylicz M., Georgopoulos C. The conserved G/F motif of the DnaJ chaperone is necessary for the activation of the substrate binding properties of the DnaK chaperone. J Biol Chem. 1995 Feb 3;270(5):2139–2144. doi: 10.1074/jbc.270.5.2139. [DOI] [PubMed] [Google Scholar]
  49. 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. Biochemistry. 1998 Jun 2;37(22):7929–7940. doi: 10.1021/bi9800855. [DOI] [PubMed] [Google Scholar]
  50. Wang T. F., Chang J. H., Wang C. Identification of the peptide binding domain of hsc70. 18-Kilodalton fragment located immediately after ATPase domain is sufficient for high affinity binding. J Biol Chem. 1993 Dec 15;268(35):26049–26051. [PubMed] [Google Scholar]
  51. Wawrzynów A., Zylicz M. 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. 1995 Aug 18;270(33):19300–19306. doi: 10.1074/jbc.270.33.19300. [DOI] [PubMed] [Google Scholar]
  52. Wishart D. S., Sykes B. D. Chemical shifts as a tool for structure determination. Methods Enzymol. 1994;239:363–392. doi: 10.1016/s0076-6879(94)39014-2. [DOI] [PubMed] [Google Scholar]
  53. Zhu X., Zhao X., Burkholder W. F., Gragerov A., Ogata C. M., Gottesman M. E., Hendrickson W. A. Structural analysis of substrate binding by the molecular chaperone DnaK. Science. 1996 Jun 14;272(5268):1606–1614. doi: 10.1126/science.272.5268.1606. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Zuiderweg E. R., Fesik S. W. Heteronuclear three-dimensional NMR spectroscopy of the inflammatory protein C5a. Biochemistry. 1989 Mar 21;28(6):2387–2391. doi: 10.1021/bi00432a008. [DOI] [PubMed] [Google Scholar]

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