Allosteric regulation of Hsp70 chaperones by a proline switch.

Crucial to the function of Hsp70 chaperones is the nucleotide-regulated transition between two conformational states, the ATP bound state with high association and dissociation rates for substrates and the ADP bound state with two and three orders of magnitude lower association and dissociation rates. The spontaneous transition between the two states is extremely slow, indicating a high energy barrier for the switch that regulates the transition. Here we provide evidence that a universally conserved proline in the ATPase domain constitutes the switch that assumes alternate conformations in response to ATP binding and hydrolysis. The conformation of the proline, acting through an invariant arginine as relay, determines and stabilizes the opened and closed conformation of the substrate binding domain and thereby regulates the chaperone activity of Hsp70.

[1]  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.

[2]  L. Gierasch,et al.  Mutations in the substrate binding domain of the Escherichia coli 70 kDa molecular chaperone, DnaK, which alter substrate affinity or interdomain coupling. , 1999, Journal of molecular biology.

[3]  W. Burkholder,et al.  Isolation and characterization of an Escherichia coli DnaK mutant with impaired ATPase activity. , 1994, Journal of molecular biology.

[4]  J. Reinstein,et al.  Nucleotide-induced Conformational Changes in the ATPase and Substrate Binding Domains of the DnaK Chaperone Provide Evidence for Interdomain Communication (*) , 1995, The Journal of Biological Chemistry.

[5]  K. Flaherty,et al.  Structural basis of the 70-kilodalton heat shock cognate protein ATP hydrolytic activity. II. Structure of the active site with ADP or ATP bound to wild type and mutant ATPase fragment. , 1994, The Journal of biological chemistry.

[6]  P. Christen,et al.  Kinetics of molecular chaperone action. , 1994, Science.

[7]  Andreas Martin,et al.  A proline switch controls folding and domain interactions in the gene-3-protein of the filamentous phage fd. , 2003, Journal of molecular biology.

[8]  E. Zuiderweg,et al.  The 70-kDa heat shock protein chaperone nucleotide-binding domain in solution unveiled as a molecular machine that can reorient its functional subdomains. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[9]  K. Wüthrich,et al.  Nmr studies of the rates of proline cis–trans isomerization in oligopeptides , 1981 .

[10]  D. Mckay,et al.  Structural replacement of active site monovalent cations by the epsilon-amino group of lysine in the ATPase fragment of bovine Hsc70. , 1998, Biochemistry.

[11]  K. Flaherty,et al.  Lysine 71 of the Chaperone Protein Hsc70 Is Essential for ATP Hydrolysis* , 1996, The Journal of Biological Chemistry.

[12]  J. Reinstein,et al.  The second step of ATP binding to DnaK induces peptide release. , 1996, Journal of molecular biology.

[13]  K. Flaherty,et al.  Three-dimensional structure of the ATPase fragment of a 70K heat-shock cognate protein , 1990, Nature.

[14]  Michael G. Sehorn,et al.  Characterization of a Lidless Form of the Molecular Chaperone DnaK , 2001, The Journal of Biological Chemistry.

[15]  D. Mckay,et al.  How Potassium Affects the Activity of the Molecular Chaperone Hsc70 , 1995, The Journal of Biological Chemistry.

[16]  F. Hartl,et al.  Molecular Chaperones in the Cytosol: from Nascent Chain to Folded Protein , 2002, Science.

[17]  C. Deluca-Flaherty,et al.  Structural basis of the 70-kilodalton heat shock cognate protein ATP hydrolytic activity. I. Kinetic analyses of active site mutants. , 1994, The Journal of biological chemistry.

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

[19]  E. Eisenberg,et al.  Auxilin-induced interaction of the molecular chaperone Hsc70 with clathrin baskets. , 1997, Biochemistry.

[20]  S. N. Witt,et al.  ATP lowers the activation enthalpy barriers to DnaK-peptide complex formation and dissociation. , 1999, Cell stress & chaperones.

[21]  The hydroxyl of threonine 13 of the bovine 70-kDa heat shock cognate protein is essential for transducing the ATP-induced conformational change. , 1998, Biochemistry.

[22]  Jonathan J Silberg,et al.  Regulation of the HscA ATPase Reaction Cycle by the Co-chaperone HscB and the Iron-Sulfur Cluster Assembly Protein IscU* , 2004, Journal of Biological Chemistry.

[23]  J. Reinstein,et al.  The role of ATP in the functional cycle of the DnaK chaperone system. , 1995, Journal of molecular biology.

[24]  A. Andreotti,et al.  Native state proline isomerization: an intrinsic molecular switch. , 2003, Biochemistry.

[25]  E. Zuiderweg,et al.  NMR Study of Nucleotide-induced Changes in the Nucleotide Binding Domain of Thermus thermophilus Hsp70 Chaperone DnaK , 2004, Journal of Biological Chemistry.

[26]  K C Holmes,et al.  A new ATP-binding fold in actin, hexokinase and Hsc70. , 1993, Trends in cell biology.

[27]  P. Christen,et al.  The power stroke of the DnaK/DnaJ/GrpE molecular chaperone system. , 1997, Journal of molecular biology.

[28]  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.

[29]  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.

[30]  Arturo Muga,et al.  Interdomain interaction through helices A and B of DnaK peptide binding domain , 2003, FEBS letters.

[31]  M. Mayer,et al.  Hsp70 chaperones: Cellular functions and molecular mechanism , 2005, Cellular and Molecular Life Sciences.

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

[33]  D. Mckay,et al.  Mapping the role of active site residues for transducing an ATP-induced conformational change in the bovine 70-kDa heat shock cognate protein. , 1999, Biochemistry.

[34]  A. Andreotti,et al.  Structural characterization of a proline-driven conformational switch within the Itk SH2 domain , 2002, Nature Structural Biology.

[35]  A. Jabs,et al.  Peptide bonds revisited , 1998, Nature Structural &Molecular Biology.

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

[37]  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.

[38]  L. Hendershot,et al.  Characterization of the Nucleotide Binding Properties and ATPase Activity of Recombinant Hamster BiP Purified from Bacteria (*) , 1995, The Journal of Biological Chemistry.

[39]  Andreas Martin,et al.  Prolyl isomerization as a molecular timer in phage infection , 2005, Nature Structural &Molecular Biology.

[40]  A. Jabs,et al.  Non-proline cis peptide bonds in proteins. , 1999, Journal of molecular biology.

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

[42]  Ulf Reimer,et al.  Barriers to Rotation of Secondary Amide Peptide Bonds , 1998 .

[43]  F. Hartl,et al.  The ATP hydrolysis-dependent reaction cycle of the Escherichia coli Hsp70 system DnaK, DnaJ, and GrpE. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[44]  B. Bukau,et al.  Mutations altering heat shock specific subunit of RNA polymerase suppress major cellular defects of E. coli mutants lacking the DnaK chaperone. , 1990, The EMBO journal.

[45]  D. Mckay,et al.  Threonine 204 of the chaperone protein Hsc70 influences the structure of the active site, but is not essential for ATP hydrolysis. , 1993, The Journal of biological chemistry.

[46]  P. Christen,et al.  Catapult mechanism renders the chaperone action of Hsp70 unidirectional. , 1998, Journal of molecular biology.

[47]  E Neher,et al.  Conductance fluctuations and ionic pores in membranes. , 1977, Annual review of biophysics and bioengineering.