Hsp90 is regulated by a switch point in the C‐terminal domain

Heat shock protein 90 (Hsp90) is an abundant, dimeric ATP‐dependent molecular chaperone, and ATPase activity is essential for its in vivo functions. S‐nitrosylation of a residue located in the carboxy‐terminal domain has been shown to affect Hsp90 activity in vivo. To understand how variation of a specific amino acid far away from the amino‐terminal ATP‐binding site regulates Hsp90 functions, we mutated the corresponding residue and analysed yeast and human Hsp90 variants both in vivo and in vitro. Here, we show that this residue is a conserved, strong regulator of Hsp90 functions, including ATP hydrolysis and chaperone activity. Unexpectedly, the variants alter both the C‐terminal and N‐terminal association properties of Hsp90, and shift its conformational equilibrium within the ATPase cycle. Thus, S‐nitrosylation of this residue allows the fast and efficient fine regulation of Hsp90.

[1]  Chrisostomos Prodromou,et al.  Regulation of Hsp90 ATPase activity by tetratricopeptide repeat (TPR)‐domain co‐chaperones , 1999, The EMBO journal.

[2]  H. Erdjument-Bromage,et al.  S-nitroso proteome of Mycobacterium tuberculosis: Enzymes of intermediary metabolism and antioxidant defense. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[3]  M. Nassal,et al.  Efficient Hsp90-independent in Vitro Activation by Hsc70 and Hsp40 of Duck Hepatitis B Virus Reverse Transcriptase, an Assumed Hsp90 Client Protein* , 2003, Journal of Biological Chemistry.

[4]  B. Freeman,et al.  p23/Sba1p Protects against Hsp90 Inhibitors Independently of Its Intrinsic Chaperone Activity , 2008, Molecular and Cellular Biology.

[5]  L. Müller,et al.  Hsp90 Regulates the Activity of Wild Type p53 under Physiological and Elevated Temperatures* , 2004, Journal of Biological Chemistry.

[6]  R. Immormino,et al.  Structures of GRP94-nucleotide complexes reveal mechanistic differences between the hsp90 chaperones. , 2007, Molecular cell.

[7]  P S Kim,et al.  Measurement of the beta-sheet-forming propensities of amino acids. , 1994, Nature.

[8]  J. Reinstein,et al.  Intrinsic Inhibition of the Hsp90 ATPase Activity* , 2006, Journal of Biological Chemistry.

[9]  P. Meyer.  Structural basis for recruitment of the ATPase activator Aha1 to the Hsp90 chaperone machinery , 2004, The EMBO journal.

[10]  J. Vázquez,et al.  S-nitrosylation of Hsp90 promotes the inhibition of its ATPase and endothelial nitric oxide synthase regulatory activities. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[11]  L. Pearl,et al.  ATP binding and hydrolysis are essential to the function of the Hsp90 molecular chaperone in vivo , 1998, The EMBO journal.

[12]  Chrisostomos Prodromou,et al.  The ATPase cycle of Hsp90 drives a molecular ‘clamp’ via transient dimerization of the N‐terminal domains , 2000, The EMBO journal.

[13]  J. Reinstein,et al.  Dissection of the Contribution of Individual Domains to the ATPase Mechanism of Hsp90* , 2003, Journal of Biological Chemistry.

[14]  N. Rosen,et al.  An acetylation site in the middle domain of Hsp90 regulates chaperone function. , 2007, Molecular cell.

[15]  Roger Fan,et al.  Dynamic activation of endothelial nitric oxide synthase by Hsp90 , 1998, Nature.

[16]  S. Lindquist,et al.  Mutational analysis of Hsp90 function: interactions with a steroid receptor and a protein kinase , 1995, Molecular and cellular biology.

[17]  References , 1971 .

[18]  David A. Agard,et al.  Structural Analysis of E. coli hsp90 Reveals Dramatic Nucleotide-Dependent Conformational Rearrangements , 2006, Cell.

[19]  A. Maxwell,et al.  The 43-kilodalton N-terminal fragment of the DNA gyrase B protein hydrolyzes ATP and binds coumarin drugs. , 1993, Biochemistry.

[20]  Jörg Durner,et al.  Proteomic Identification of S-Nitrosylated Proteins in Arabidopsis1[w] , 2005, Plant Physiology.

[21]  J. Johnson,et al.  Characterization of a novel 23-kilodalton protein of unactive progesterone receptor complexes , 1994, Molecular and cellular biology.

[22]  S. Lindquist,et al.  HSP90 and the chaperoning of cancer , 2005, Nature Reviews Cancer.

[23]  J. Buchner,et al.  The phosphatase Ppt1 is a dedicated regulator of the molecular chaperone Hsp90 , 2006, The EMBO journal.

[24]  Paul Workman,et al.  Activation of the ATPase activity of hsp90 by the stress-regulated cochaperone aha1. , 2002, Molecular cell.

[25]  J. M. Gallardo,et al.  High-sensitivity analysis of specific peptides in complex samples by selected MS/MS ion monitoring and linear ion trap mass spectrometry: application to biological studies. , 2007, Journal of mass spectrometry : JMS.

[26]  L. Pearl,et al.  Crystal structure of an Hsp90–nucleotide–p23/Sba1 closed chaperone complex , 2006, Nature.

[27]  J. Reinstein,et al.  Conserved Conformational Changes in the ATPase Cycle of Human Hsp90* , 2008, Journal of Biological Chemistry.

[28]  Johannes Buchner,et al.  The Co-chaperone Sba1 connects the ATPase reaction of Hsp90 to the progression of the chaperone cycle. , 2004, Journal of molecular biology.

[29]  F. Hartl,et al.  In Vivo Function of Hsp90 Is Dependent on ATP Binding and ATP Hydrolysis , 1998, The Journal of cell biology.

[30]  D. Picard,et al.  Heat-shock protein 90, a chaperone for folding and regulation , 2002, Cellular and Molecular Life Sciences CMLS.

[31]  M. Mayer,et al.  Spatially and kinetically resolved changes in the conformational dynamics of the Hsp90 chaperone machine , 2009, The EMBO journal.

[32]  Johannes Buchner,et al.  The ATPase Cycle of the Endoplasmic Chaperone Grp94* , 2007, Journal of Biological Chemistry.

[33]  M. Retzlaff,et al.  Chaperone activation of the hepadnaviral reverse transcriptase for template RNA binding is established by the Hsp70 and stimulated by the Hsp90 system , 2007, Nucleic acids research.

[34]  Giorgio Colombo,et al.  Modeling Signal Propagation Mechanisms and Ligand-Based Conformational Dynamics of the Hsp90 Molecular Chaperone Full-Length Dimer , 2009, PLoS Comput. Biol..

[35]  J. Reinstein,et al.  Sti1 Is a Non-competitive Inhibitor of the Hsp90 ATPase , 2003, The Journal of Biological Chemistry.

[36]  Marco Siderius,et al.  The Middle Domain of Hsp90 Acts as a Discriminator between Different Types of Client Proteins , 2006, Molecular and Cellular Biology.

[37]  Martin Hessling,et al.  Dissection of the ATP-induced conformational cycle of the molecular chaperone Hsp90 , 2009, Nature Structural &Molecular Biology.

[38]  Jason C. Young,et al.  Polypeptide release by Hsp90 involves ATP hydrolysis and is enhanced by the co‐chaperone p23 , 2000, The EMBO journal.

[39]  J. Buchner,et al.  Coordinated ATP Hydrolysis by the Hsp90 Dimer* , 2001, The Journal of Biological Chemistry.

[40]  J. Reinstein,et al.  The ATPase Cycle of the Mitochondrial Hsp90 Analog Trap1* , 2008, Journal of Biological Chemistry.

[41]  J. Boros,et al.  Hsp90 Chaperones Wild-type p53 Tumor Suppressor Protein* , 2004, Journal of Biological Chemistry.