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. 2002 Nov;162(3):1045–1053. doi: 10.1093/genetics/162.3.1045

Scanning mutagenesis identifies amino acid residues essential for the in vivo activity of the Escherichia coli DnaJ (Hsp40) J-domain.

Pierre Genevaux 1, Françoise Schwager 1, Costa Georgopoulos 1, William L Kelley 1
PMCID: PMC1462316  PMID: 12454054

Abstract

The DnaJ (Hsp40) cochaperone regulates the DnaK (Hsp70) chaperone by accelerating ATP hydrolysis in a cycle closely linked to substrate binding and release. The J-domain, the signature motif of the Hsp40 family, orchestrates interaction with the DnaK ATPase domain. We studied the J-domain by creating 42 mutant E. coli DnaJ variants and examining their phenotypes in various separate in vivo assays, namely, bacterial growth at low and high temperatures, motility, and propagation of bacteriophage lambda. Most mutants studied behaved like wild type in all assays. In addition to the (33)HisProAsp(35) (HPD) tripeptide found in all known functional J-domains, our study uncovered three new single substitution mutations (Y25A, K26A, and F47A) that totally abolish J-domain function. Furthermore, two glycine substitution mutants in an exposed flexible loop (R36G, N37G) showed partial loss of J-domain function alone and complete loss of function as a triple (RNQ-GGG) mutant coupled with the phenotypically silent Q38G. Interestingly, all the essential residues map to a small region on the same solvent-exposed face of the J-domain. Engineered mutations in the corresponding residues of the human Hdj1 J-domain grafted in E. coli DnaJ also resulted in loss of function, suggesting an evolutionarily conserved interaction surface. We propose that these clustered residues impart critical sequence determinants necessary for J-domain catalytic activity and reversible contact interface with the DnaK ATPase domain.

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Selected References

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  1. Auger I., Escola J. M., Gorvel J. P., Roudier J. HLA-DR4 and HLA-DR10 motifs that carry susceptibility to rheumatoid arthritis bind 70-kD heat shock proteins. Nat Med. 1996 Mar;2(3):306–310. doi: 10.1038/nm0396-306. [DOI] [PubMed] [Google Scholar]
  2. Berjanskii M. V., Riley M. I., Xie A., Semenchenko V., Folk W. R., Van Doren S. R. NMR structure of the N-terminal J domain of murine polyomavirus T antigens. Implications for DnaJ-like domains and for mutations of T antigens. J Biol Chem. 2000 Nov 17;275(46):36094–36103. doi: 10.1074/jbc.M006572200. [DOI] [PubMed] [Google Scholar]
  3. Bukau B., Horwich A. L. The Hsp70 and Hsp60 chaperone machines. Cell. 1998 Feb 6;92(3):351–366. doi: 10.1016/s0092-8674(00)80928-9. [DOI] [PubMed] [Google Scholar]
  4. Cyr D. M., Lu X., Douglas M. G. Regulation of Hsp70 function by a eukaryotic DnaJ homolog. J Biol Chem. 1992 Oct 15;267(29):20927–20931. [PubMed] [Google Scholar]
  5. Deloche O., Kelley W. L., Georgopoulos C. Structure-function analyses of the Ssc1p, Mdj1p, and Mge1p Saccharomyces cerevisiae mitochondrial proteins in Escherichia coli. J Bacteriol. 1997 Oct;179(19):6066–6075. doi: 10.1128/jb.179.19.6066-6075.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Gassler C. S., Wiederkehr T., Brehmer D., Bukau B., Mayer M. P. Bag-1M accelerates nucleotide release for human Hsc70 and Hsp70 and can act concentration-dependent as positive and negative cofactor. J Biol Chem. 2001 Jul 5;276(35):32538–32544. doi: 10.1074/jbc.M105328200. [DOI] [PubMed] [Google Scholar]
  7. Genevaux P., Wawrzynow A., Zylicz M., Georgopoulos C., Kelley W. L. DjlA is a third DnaK co-chaperone of Escherichia coli, and DjlA-mediated induction of colanic acid capsule requires DjlA-DnaK interaction. J Biol Chem. 2000 Dec 5;276(11):7906–7912. doi: 10.1074/jbc.M003855200. [DOI] [PubMed] [Google Scholar]
  8. Greene M. K., Maskos K., Landry S. J. Role of the J-domain in the cooperation of Hsp40 with Hsp70. Proc Natl Acad Sci U S A. 1998 May 26;95(11):6108–6113. doi: 10.1073/pnas.95.11.6108. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Gässler C. S., Buchberger A., Laufen T., Mayer M. P., Schröder H., Valencia A., Bukau B. Mutations in the DnaK chaperone affecting interaction with the DnaJ cochaperone. Proc Natl Acad Sci U S A. 1998 Dec 22;95(26):15229–15234. doi: 10.1073/pnas.95.26.15229. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Hartl F. U. Molecular chaperones in cellular protein folding. Nature. 1996 Jun 13;381(6583):571–579. doi: 10.1038/381571a0. [DOI] [PubMed] [Google Scholar]
  11. Hennessy F., Cheetham M. E., Dirr H. W., Blatch G. L. Analysis of the levels of conservation of the J domain among the various types of DnaJ-like proteins. Cell Stress Chaperones. 2000 Oct;5(4):347–358. doi: 10.1379/1466-1268(2000)005<0347:aotloc>2.0.co;2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Higuchi R., Krummel B., Saiki R. K. A general method of in vitro preparation and specific mutagenesis of DNA fragments: study of protein and DNA interactions. Nucleic Acids Res. 1988 Aug 11;16(15):7351–7367. doi: 10.1093/nar/16.15.7351. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Huang K., Ghose R., Flanagan J. M., Prestegard J. H. Backbone dynamics of the N-terminal domain in E. coli DnaJ determined by 15N- and 13CO-relaxation measurements. Biochemistry. 1999 Aug 10;38(32):10567–10577. doi: 10.1021/bi990263+. [DOI] [PubMed] [Google Scholar]
  14. 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]
  15. Kelley W. L., Georgopoulos C. The T/t common exon of simian virus 40, JC, and BK polyomavirus T antigens can functionally replace the J-domain of the Escherichia coli DnaJ molecular chaperone. Proc Natl Acad Sci U S A. 1997 Apr 15;94(8):3679–3684. doi: 10.1073/pnas.94.8.3679. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Kelley W. L. The J-domain family and the recruitment of chaperone power. Trends Biochem Sci. 1998 Jun;23(6):222–227. doi: 10.1016/s0968-0004(98)01215-8. [DOI] [PubMed] [Google Scholar]
  17. Kim H. Y., Ahn B. Y., Cho Y. Structural basis for the inactivation of retinoblastoma tumor suppressor by SV40 large T antigen. EMBO J. 2001 Jan 15;20(1-2):295–304. doi: 10.1093/emboj/20.1.295. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Kunkel T. A., Roberts J. D., Zakour R. A. Rapid and efficient site-specific mutagenesis without phenotypic selection. Methods Enzymol. 1987;154:367–382. doi: 10.1016/0076-6879(87)54085-x. [DOI] [PubMed] [Google Scholar]
  19. 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]
  20. Liberek K., Wall D., Georgopoulos C. The DnaJ chaperone catalytically activates the DnaK chaperone to preferentially bind the sigma 32 heat shock transcriptional regulator. Proc Natl Acad Sci U S A. 1995 Jul 3;92(14):6224–6228. doi: 10.1073/pnas.92.14.6224. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Lyman S. K., Schekman R. Binding of secretory precursor polypeptides to a translocon subcomplex is regulated by BiP. Cell. 1997 Jan 10;88(1):85–96. doi: 10.1016/s0092-8674(00)81861-9. [DOI] [PubMed] [Google Scholar]
  22. Misselwitz B., Staeck O., Rapoport T. A. J proteins catalytically activate Hsp70 molecules to trap a wide range of peptide sequences. Mol Cell. 1998 Nov;2(5):593–603. doi: 10.1016/s1097-2765(00)80158-6. [DOI] [PubMed] [Google Scholar]
  23. Peden K. W., Pipas J. M. Simian virus 40 mutants with amino-acid substitutions near the amino terminus of large T antigen. Virus Genes. 1992 Apr;6(2):107–118. doi: 10.1007/BF01703060. [DOI] [PubMed] [Google Scholar]
  24. 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]
  25. Qian Y. Q., Patel D., Hartl F. U., McColl D. J. Nuclear magnetic resonance solution structure of the human Hsp40 (HDJ-1) J-domain. J Mol Biol. 1996 Jul 12;260(2):224–235. doi: 10.1006/jmbi.1996.0394. [DOI] [PubMed] [Google Scholar]
  26. Schlenstedt G., Harris S., Risse B., Lill R., Silver P. A. A yeast DnaJ homologue, Scj1p, can function in the endoplasmic reticulum with BiP/Kar2p via a conserved domain that specifies interactions with Hsp70s. J Cell Biol. 1995 May;129(4):979–988. doi: 10.1083/jcb.129.4.979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Shi W., Zhou Y., Wild J., Adler J., Gross C. A. DnaK, DnaJ, and GrpE are required for flagellum synthesis in Escherichia coli. J Bacteriol. 1992 Oct;174(19):6256–6263. doi: 10.1128/jb.174.19.6256-6263.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Suh W. C., Burkholder W. F., Lu C. Z., Zhao X., Gottesman M. E., Gross C. A. Interaction of the Hsp70 molecular chaperone, DnaK, with its cochaperone DnaJ. Proc Natl Acad Sci U S A. 1998 Dec 22;95(26):15223–15228. doi: 10.1073/pnas.95.26.15223. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Sullivan C. S., Tremblay J. D., Fewell S. W., Lewis J. A., Brodsky J. L., Pipas J. M. Species-specific elements in the large T-antigen J domain are required for cellular transformation and DNA replication by simian virus 40. Mol Cell Biol. 2000 Aug;20(15):5749–5757. doi: 10.1128/mcb.20.15.5749-5757.2000. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Szabo A., Langer T., Schröder H., Flanagan J., Bukau B., Hartl F. U. The ATP hydrolysis-dependent reaction cycle of the Escherichia coli Hsp70 system DnaK, DnaJ, and GrpE. Proc Natl Acad Sci U S A. 1994 Oct 25;91(22):10345–10349. doi: 10.1073/pnas.91.22.10345. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. 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]
  32. Tsai J., Douglas M. G. A conserved HPD sequence of the J-domain is necessary for YDJ1 stimulation of Hsp70 ATPase activity at a site distinct from substrate binding. J Biol Chem. 1996 Apr 19;271(16):9347–9354. doi: 10.1074/jbc.271.16.9347. [DOI] [PubMed] [Google Scholar]
  33. Ueguchi C., Shiozawa T., Kakeda M., Yamada H., Mizuno T. A study of the double mutation of dnaJ and cbpA, whose gene products function as molecular chaperones in Escherichia coli. J Bacteriol. 1995 Jul;177(13):3894–3896. doi: 10.1128/jb.177.13.3894-3896.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Wall D., Zylicz M., Georgopoulos C. The NH2-terminal 108 amino acids of the Escherichia coli DnaJ protein stimulate the ATPase activity of DnaK and are sufficient for lambda replication. J Biol Chem. 1994 Feb 18;269(7):5446–5451. [PubMed] [Google Scholar]
  35. Wild J., Altman E., Yura T., Gross C. A. DnaK and DnaJ heat shock proteins participate in protein export in Escherichia coli. Genes Dev. 1992 Jul;6(7):1165–1172. doi: 10.1101/gad.6.7.1165. [DOI] [PubMed] [Google Scholar]
  36. Ziegelhoffer T., Lopez-Buesa P., Craig E. A. The dissociation of ATP from hsp70 of Saccharomyces cerevisiae is stimulated by both Ydj1p and peptide substrates. J Biol Chem. 1995 May 5;270(18):10412–10419. doi: 10.1074/jbc.270.18.10412. [DOI] [PubMed] [Google Scholar]

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