Molecular basis for regulation of the heat shock transcription factor sigma32 by the DnaK and DnaJ chaperones.

Central to the transcriptional control of the Escherichia coli heat shock regulon is the stress-dependent inhibition of the sigma(32) subunit of RNA polymerase by reversible association with the DnaK chaperone, mediated by the DnaJ cochaperone. Here we identified two distinct sites in sigma(32) as binding sites for DnaK and DnaJ. DnaJ binding destabilizes a distant region of sigma(32) in close spatial vicinity of the DnaK-binding site, and DnaK destabilizes a region in the N-terminal domain, the primary target for the FtsH protease, which degrades sigma(32) in vivo. Our findings suggest a molecular mechanism for the DnaK- and DnaJ-mediated inactivation of sigma(32) as part of the heat shock response. They furthermore demonstrate that DnaK and DnaJ binding can induce conformational changes in a native protein substrate even at distant sites, a feature that we propose to be of general relevance for the action of Hsp70 chaperone systems.

[1]  C. Georgopoulos,et al.  Modulation of stability of the Escherichia coli heat shock regulatory factor sigma , 1989, Journal of bacteriology.

[2]  H. Bujard,et al.  Physical interaction between heat shock proteins DnaK, DnaJ, and GrpE and the bacterial heat shock transcription factor σ 32 , 1992, Cell.

[3]  A. Karzai,et al.  A Bipartite Signaling Mechanism Involved in DnaJ-mediated Activation of the Escherichia coli DnaK Protein (*) , 1996, The Journal of Biological Chemistry.

[4]  P. Christen,et al.  Mechanism of the Targeting Action of DnaJ in the DnaK Molecular Chaperone System* , 2003, Journal of Biological Chemistry.

[5]  C. Gross,et al.  Analysis of sigma32 mutants defective in chaperone-mediated feedback control reveals unexpected complexity of the heat shock response. , 2007, Proceedings of the National Academy of Sciences of the United States of America.

[6]  Bernd Bukau,et al.  Multistep mechanism of substrate binding determines chaperone activity of Hsp70 , 2000, Nature Structural Biology.

[7]  A. Valencia,et al.  A conserved loop in the ATPase domain of the DnaK chaperone is essential for stable binding of GrpE , 1994, Nature Structural Biology.

[8]  P. Roepstorff,et al.  Mapping Temperature-induced Conformational Changes in the Escherichia coli Heat Shock Transcription Factor σ32 by Amide Hydrogen Exchange* , 2003, Journal of Biological Chemistry.

[9]  Craig M. Ogata,et al.  Structural Analysis of Substrate Binding by the Molecular Chaperone DnaK , 1996, Science.

[10]  H. Yanagi,et al.  Marked instability of the sigma(32) heat shock transcription factor at high temperature. Implications for heat shock regulation. , 1999, The Journal of biological chemistry.

[11]  A. Fersht,et al.  Alpha-helix stability in proteins. II. Factors that influence stability at an internal position. , 1992, Journal of molecular biology.

[12]  T. Ogura,et al.  Spectrometric analysis of degradation of a physiological substrate sigma32 by Escherichia coli AAA protease FtsH. , 2004, Journal of structural biology.

[13]  H. Mori,et al.  Escherichia coli FtsH is a membrane‐bound, ATP‐dependent protease which degrades the heat‐shock transcription factor sigma 32. , 1995, The EMBO journal.

[14]  Hirotada Mori,et al.  Heat shock regulation in the ftsH null mutant of Escherichia coli: dissection of stability and activity control mechanisms of σ32in vivo , 1998, Molecular microbiology.

[15]  M. Zulauf,et al.  Investigation of the molecular chaperone DnaJ by analytical ultracentrifugation , 1995 .

[16]  C. Gross,et al.  Analysis of σ32 mutants defective in chaperone-mediated feedback control reveals unexpected complexity of the heat shock response , 2007, Proceedings of the National Academy of Sciences.

[17]  M C Peitsch,et al.  ProMod and Swiss-Model: Internet-based tools for automated comparative protein modelling. , 1996, Biochemical Society transactions.

[18]  F. Narberhaus,et al.  Identification of a Turnover Element in Region 2.1 of Escherichia coli σ32 by a Bacterial One-Hybrid Approach , 2005, Journal of bacteriology.

[19]  Bernd Bukau,et al.  Substrate specificity of the DnaK chaperone determined by screening cellulose‐bound peptide libraries , 1997, The EMBO journal.

[20]  F. Neidhart.  Escherichia coli and Salmonella. , 1996 .

[21]  M. Mayer,et al.  Investigation of the interaction between DnaK and DnaJ by surface plasmon resonance spectroscopy. , 1999, Journal of molecular biology.

[22]  C. Gross,et al.  The heat shock response of E. coli is regulated by changes in the concentration of sigma 32. , 1987, Nature.

[23]  V. de Lorenzo,et al.  The black cat/white cat principle of signal integration in bacterial promoters , 2001 .

[24]  S. Darst,et al.  Disulfide cross-linking indicates that FlgM-bound and free σ28 adopt similar conformations , 2006, Proceedings of the National Academy of Sciences.

[25]  Seth A. Darst,et al.  Crystal Structure of the Flagellar σ/Anti-σ Complex σ28/FlgM Reveals an Intact σ Factor in an Inactive Conformation , 2004 .

[26]  C. Gross,et al.  DnaK, DnaJ, and GrpE heat shock proteins negatively regulate heat shock gene expression by controlling the synthesis and stability of sigma 32. , 1990, Genes & development.

[27]  H. Bujard,et al.  A cycle of binding and release of the DnaK, DnaJ and GrpE chaperones regulates activity of the Escherichia coli heat shock transcription factor sigma32. , 1996, The EMBO journal.

[28]  R. Burgess,et al.  Purification and properties of the sigma subunit of Escherichia coli DNA-dependent RNA polymerase. , 1979, Biochemistry.

[29]  U. K. Laemmli,et al.  Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4 , 1970, Nature.

[30]  B. Matthews,et al.  Structural basis of amino acid alpha helix propensity. , 1993, Science.

[31]  Jingzhi Li,et al.  The crystal structure of the yeast Hsp40 Ydj1 complexed with its peptide substrate. , 2003, Structure.

[32]  J. Hoskins,et al.  Interaction of the DnaK and DnaJ Chaperone System with a Native Substrate, P1 RepA* , 2002, The Journal of Biological Chemistry.

[33]  Carol A. Gross,et al.  The heat shock response of E. coli is regulated by changes in the concentration of σ32 , 1987, Nature.

[34]  W. DeGrado,et al.  A thermodynamic scale for the helix-forming tendencies of the commonly occurring amino acids. , 1990, Science.

[35]  C. Gross,et al.  Lack of a robust unfoldase activity confers a unique level of substrate specificity to the universal AAA protease FtsH. , 2003, Molecular cell.

[36]  T. Galitski,et al.  The DnaK chaperone modulates the heat shock response of Escherichia coli by binding to the sigma 32 transcription factor. , 1992, Proceedings of the National Academy of Sciences of the United States of America.

[37]  B. Bukau,et al.  Role of Region C in Regulation of the Heat Shock Gene-Specific Sigma Factor of Escherichia coli, ς32 , 1999, Journal of bacteriology.

[38]  A. Grossman,et al.  Sigma 32 synthesis can regulate the synthesis of heat shock proteins in Escherichia coli. , 1987, Genes & development.

[39]  S. Yokoyama,et al.  Crystal structure of a bacterial RNA polymerase holoenzyme at 2.6 Å resolution , 2002, Nature.

[40]  C. Georgopoulos,et al.  The dnaK protein modulates the heat-shock response of Escherichia coli , 1983, Cell.

[41]  P. Bouloc,et al.  Degradation of sigma 32, the heat shock regulator in Escherichia coli, is governed by HflB. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[42]  K. Nakahigashi,et al.  Regulation of the heat-shock response. , 1999, Current opinion in microbiology.

[43]  B. Bukau.  Regulation of the Escherichia coli heat‐shock response , 1993, Molecular microbiology.

[44]  J. Schneider-Mergener,et al.  Regulatory region C of the E. coli heat shock transcription factor, sigma32, constitutes a DnaK binding site and is conserved among eubacteria. , 1996, Journal of molecular biology.

[45]  J. Reinstein,et al.  Mechanism of regulation of hsp70 chaperones by DnaJ cochaperones. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[46]  C. Georgopoulos,et al.  The DnaJ chaperone catalytically activates the DnaK chaperone to preferentially bind the sigma 32 heat shock transcriptional regulator. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[47]  S. Darst,et al.  Crystal Structure of the Bacillus stearothermophilus Anti-σ Factor SpoIIAB with the Sporulation σ Factor σF , 2002, Cell.

[48]  S. Darst,et al.  Crystal structure of the Bacillus stearothermophilus anti-sigma factor SpoIIAB with the sporulation sigma factor sigmaF. , 2002, Cell.

[49]  T. Kunkel,et al.  Efficient site-directed mutagenesis using uracil-containing DNA. , 1991, Methods in enzymology.

[50]  S. Ray,et al.  Crystal structure of the flagellar sigma/anti-sigma complex sigma(28)/FlgM reveals an intact sigma factor in an inactive conformation. , 2004, Molecular cell.

[51]  L. J. Perry,et al.  Disulfide bonds and thermal stability in T4 lysozyme. , 1988, Proceedings of the National Academy of Sciences of the United States of America.

[52]  M. Mayer,et al.  Analysis of subsecond protein dynamics by amide hydrogen exchange and mass spectrometry using a quenched‐flow setup , 2005, Protein science : a publication of the Protein Society.

[53]  A. Fersht,et al.  Engineered disulfide bonds as probes of the folding pathway of barnase: increasing the stability of proteins against the rate of denaturation. , 1993, Biochemistry.

[54]  C. Gross,et al.  Crystal Structure of Escherichia coli σE with the Cytoplasmic Domain of Its Anti-σ RseA , 2003 .

[55]  Bernd Bukau,et al.  Its substrate specificity characterizes the DnaJ co‐chaperone as a scanning factor for the DnaK chaperone , 2001, The EMBO journal.

[56]  K. Resing,et al.  Protein analysis by hydrogen exchange mass spectrometry. , 2003, Annual review of biophysics and biomolecular structure.