GR chaperone cycle mechanism revealed by cryo-EM: inactivation of GR by GR:Hsp90:Hsp70:Hop client-loading complex

Maintaining a healthy proteome is fundamental for organism survival1,2. Integral to this are Hsp90 and Hsp70 molecular chaperones that together facilitate the folding, remodeling and maturation of Hsp90’s many “client” proteins3–7. The glucocorticoid receptor (GR) is a model client strictly dependent upon Hsp90/Hsp70 for activity8–13. Chaperoning GR involves a cycle of inactivation by Hsp70, formation of an inactive GR:Hsp90:Hsp70:Hop “loading” complex, conversion to an active GR:Hsp90:p23 “maturation” complex, and subsequent GR release14. Unfortunately, a molecular understanding of this intricate chaperone cycle is lacking for any client. Here, we report the cryo-EM structure of the GR loading complex, in which Hsp70 loads GR onto Hsp90, revealing the molecular basis of direct Hsp90/Hsp70 coordination. The structure reveals two Hsp70s—one delivering GR and the other scaffolding Hop. Unexpectedly, the Hop cochaperone interacts with all components of the complex including GR, poising Hsp90 for subsequent ATP hydrolysis. GR is partially unfolded and recognized via an extended binding pocket composed of Hsp90, Hsp70 and Hop, revealing the mechanism of GR loading and inactivation. Together with the GR maturation complex (Noddings et al., 2020), we present the first complete molecular mechanism of chaperone-dependent client remodeling, establishing general principles of client recognition, inhibition, transfer and activation.

[1]  D. Elnatan,et al.  Cryo-EM structures reveal a multistep mechanism of Hsp90 activation by co-chaperone Aha1 , 2020, bioRxiv.

[2]  D. Agard,et al.  The interplay between Bag-1, Hsp70, and Hsp90 reveals that inhibiting Hsp70 rebinding is essential for Glucocorticoid Receptor activity , 2020, bioRxiv.

[3]  D. Bolon,et al.  Comprehensive fitness maps of Hsp90 show widespread environmental dependence , 2020, eLife.

[4]  M B B Gutierres,et al.  ChaperISM: improved chaperone binding prediction using position-independent scoring matrices , 2019, Bioinform..

[5]  J. Hoskins,et al.  Intermolecular Interactions between Hsp90 and Hsp70. , 2019, Journal of molecular biology.

[6]  Nadinath B. Nillegoda,et al.  The Hsp70 chaperone network , 2019, Nature Reviews Molecular Cell Biology.

[7]  D. Lamb,et al.  Coordinated Conformational Processing of the Tumor Suppressor Protein p53 by the Hsp70 and Hsp90 Chaperone Machineries. , 2019, Molecular cell.

[8]  M. Boysen,et al.  Hsp70- and Hsp90-Mediated Regulation of the Conformation of p53 DNA Binding Domain and p53 Cancer Variants. , 2019, Molecular cell.

[9]  Ming Sun,et al.  The endoplasmic reticulum (ER) chaperones BiP and Grp94 selectively associate when BiP is in the ADP conformation , 2019, The Journal of Biological Chemistry.

[10]  M. Mayer,et al.  The Hsp70-Hsp90 Chaperone Cascade in Protein Folding. , 2019, Trends in cell biology.

[11]  L. Gierasch,et al.  Recent advances in the structural and mechanistic aspects of Hsp70 molecular chaperones , 2018, The Journal of Biological Chemistry.

[12]  S. Wickner,et al.  Hsp90 and Hsp70 chaperones: Collaborators in protein remodeling , 2018, The Journal of Biological Chemistry.

[13]  Johannes Buchner,et al.  Single-molecule force spectroscopy reveals folding steps associated with hormone binding and activation of the glucocorticoid receptor , 2018, Proceedings of the National Academy of Sciences.

[14]  Isabella Haberbosch,et al.  Software tools for automated transmission electron microscopy , 2018, Nature Methods.

[15]  Shailesh Kumar,et al.  Dual Roles for Yeast Sti1/Hop in Regulating the Hsp90 Chaperone Cycle , 2018, Genetics.

[16]  H. Tadakuma,et al.  Conformational Activation of Argonaute by Distinct yet Coordinated Actions of the Hsp70 and Hsp90 Chaperone Systems. , 2018, Molecular cell.

[17]  Roman Kityk,et al.  Hsp90 Breaks the Deadlock of the Hsp70 Chaperone System. , 2018, Molecular cell.

[18]  Ville R. I. Kaila,et al.  A switch point in the molecular chaperone Hsp90 responding to client interaction , 2018, Nature Communications.

[19]  J. Hoskins,et al.  Functional and physical interaction between yeast Hsp90 and Hsp70 , 2018, Proceedings of the National Academy of Sciences.

[20]  Roman Kityk,et al.  Molecular Mechanism of J-Domain-Triggered ATP Hydrolysis by Hsp70 Chaperones. , 2017, Molecular cell.

[21]  J. Buchner,et al.  The Plasticity of the Hsp90 Co-chaperone System. , 2017, Molecular cell.

[22]  Wing-Yiu Choy,et al.  The Hsp70/Hsp90 Chaperone Machinery in Neurodegenerative Diseases , 2017, Front. Neurosci..

[23]  J. Buchner,et al.  The HSP90 chaperone machinery , 2017, Nature Reviews Molecular Cell Biology.

[24]  D. Agard,et al.  MotionCor2: anisotropic correction of beam-induced motion for improved cryo-electron microscopy , 2017, Nature Methods.

[25]  J. Buchner,et al.  BiPPred: Combined sequence‐ and structure‐based prediction of peptide binding to the Hsp70 chaperone BiP , 2016, Proteins.

[26]  J. Kim,et al.  2.4 Å resolution crystal structure of human TRAP1NM, the Hsp90 paralog in the mitochondrial matrix. , 2016, Acta crystallographica. Section D, Structural biology.

[27]  D. Agard,et al.  Atomic structure of Hsp90-Cdc37-Cdk4 reveals that Hsp90 traps and stabilizes an unfolded kinase , 2016, Science.

[28]  Frank DiMaio,et al.  Automated structure refinement of macromolecular assemblies from cryo-EM maps using Rosetta , 2016, bioRxiv.

[29]  J. Hoskins,et al.  Hsp70 and Hsp90 of E. coli Directly Interact for Collaboration in Protein Remodeling. , 2015, Journal of molecular biology.

[30]  N. Grigorieff,et al.  CTFFIND4: Fast and accurate defocus estimation from electron micrographs , 2015, bioRxiv.

[31]  R. Morimoto,et al.  The biology of proteostasis in aging and disease. , 2015, Annual review of biochemistry.

[32]  D. Agard,et al.  Hsp70 Forms Antiparallel Dimers Stabilized by Post-translational Modifications to Position Clients for Transfer to Hsp90 , 2015, Cell reports.

[33]  D. Lamb,et al.  Hsp90 regulates the dynamics of its cochaperone Sti1 and the transfer of Hsp70 between modules , 2015, Nature Communications.

[34]  A. Bracher,et al.  The nucleotide exchange factors of Hsp70 molecular chaperones , 2015, Front. Mol. Biosci..

[35]  J. Gestwicki,et al.  Targeting Hsp90/Hsp70-based protein quality control for treatment of adult onset neurodegenerative diseases. , 2015, Annual review of pharmacology and toxicology.

[36]  M. Hipp,et al.  Proteostasis impairment in protein-misfolding and -aggregation diseases. , 2014, Trends in cell biology.

[37]  R. Marmorstein,et al.  Crystal Structure of the Stress-Inducible Human Heat Shock Protein 70 Substrate-Binding Domain in Complex with Peptide Substrate , 2014, PloS one.

[38]  Bonnie Berger,et al.  A Quantitative Chaperone Interaction Network Reveals the Architecture of Cellular Protein Homeostasis Pathways , 2014, Cell.

[39]  D. Agard,et al.  Glucocorticoid Receptor Function Regulated by Coordinated Action of the Hsp90 and Hsp70 Chaperone Cycles , 2014, Cell.

[40]  J. Buchner,et al.  Modulation of the Hsp90 chaperone cycle by a stringent client protein. , 2014, Molecular cell.

[41]  David Baker,et al.  High-resolution comparative modeling with RosettaCM. , 2013, Structure.

[42]  R. Hoffmann,et al.  Structural studies on the forward and reverse binding modes of peptides to the chaperone DnaK. , 2013, Journal of molecular biology.

[43]  Yujin E. Kim,et al.  Molecular chaperone functions in protein folding and proteostasis. , 2013, Annual review of biochemistry.

[44]  W. Chiu,et al.  Cryo‐EM model validation using independent map reconstructions , 2013, Protein science : a publication of the Protein Society.

[45]  Qun Liu,et al.  Allosteric opening of the polypeptide-binding site when an Hsp70 binds ATP , 2013, Nature Structural &Molecular Biology.

[46]  D. Agard,et al.  Uncovering a region of heat shock protein 90 important for client binding in E. coli and chaperone function in yeast. , 2013, Molecular cell.

[47]  Roman Kityk,et al.  Structure and dynamics of the ATP-bound open conformation of Hsp70 chaperones. , 2012, Molecular cell.

[48]  Lila M. Gierasch,et al.  An Interdomain Energetic Tug-of-War Creates the Allosterically Active State in Hsp70 Molecular Chaperones , 2012, Cell.

[49]  S. Scheres.  RELION: Implementation of a Bayesian approach to cryo-EM structure determination , 2012, Journal of structural biology.

[50]  Susan Lindquist,et al.  Quantitative Analysis of Hsp90-Client Interactions Reveals Principles of Substrate Recognition , 2012, Cell.

[51]  Yechun Xu,et al.  Structure insights into mechanisms of ATP hydrolysis and the activation of human heat-shock protein 90. , 2012, Acta biochimica et biophysica Sinica.

[52]  J. Buchner,et al.  The architecture of functional modules in the Hsp90 co‐chaperone Sti1/Hop , 2012, The EMBO journal.

[53]  M. Mayer,et al.  Dynamics of the regulation of Hsp90 by the co‐chaperone Sti1 , 2012, The EMBO journal.

[54]  Shigeyuki Yokoyama,et al.  Biochemical and structural studies on the high affinity of Hsp70 for ADP , 2011, Protein science : a publication of the Protein Society.

[55]  D. Agard,et al.  Client-loading conformation of the Hsp90 molecular chaperone revealed in the cryo-EM structure of the human Hsp90:Hop complex. , 2011, Molecular cell.

[56]  J. Hoskins,et al.  Heat shock protein 90 from Escherichia coli collaborates with the DnaK chaperone system in client protein remodeling , 2011, Proceedings of the National Academy of Sciences.

[57]  D. Agard,et al.  Substrate binding drives large-scale conformational changes in the Hsp90 molecular chaperone. , 2011, Molecular cell.

[58]  David A Agard,et al.  Conformational dynamics of the molecular chaperone Hsp90 , 2011, Quarterly Reviews of Biophysics.

[59]  G. Giaccone,et al.  Targeting the dynamic HSP90 complex in cancer , 2010, Nature Reviews Cancer.

[60]  S. Lindquist,et al.  HSP90 at the hub of protein homeostasis: emerging mechanistic insights , 2010, Nature Reviews Molecular Cell Biology.

[61]  E. Zuiderweg,et al.  Solution conformation of wild-type E. coli Hsp70 (DnaK) chaperone complexed with ADP and substrate , 2009, Proceedings of the National Academy of Sciences.

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

[63]  D. Toft,et al.  Minireview: the intersection of steroid receptors with molecular chaperones: observations and questions. , 2008, Molecular endocrinology.

[64]  Lila M Gierasch,et al.  Hsp70 chaperone ligands control domain association via an allosteric mechanism mediated by the interdomain linker. , 2007, Molecular cell.

[65]  Jill L. Johnson,et al.  Nucleotide-Dependent Interaction of Saccharomyces cerevisiae Hsp90 with the Cochaperone Proteins Sti1, Cpr6, and Sba1 , 2006, Molecular and Cellular Biology.

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

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

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

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

[70]  Johannes Söding,et al.  The HHpred interactive server for protein homology detection and structure prediction , 2005, Nucleic Acids Res..

[71]  Johannes Buchner,et al.  Sti1 Is a Novel Activator of the Ssa Proteins* , 2003, Journal of Biological Chemistry.

[72]  Chrisostomos Prodromou,et al.  Structural and functional analysis of the middle segment of hsp90: implications for ATP hydrolysis and client protein and cochaperone interactions. , 2003, Molecular cell.

[73]  Andrew B. Martin,et al.  Addition of a photocrosslinking amino acid to the genetic code of Escherichia coli , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[74]  T. Willson,et al.  Crystal Structure of the Glucocorticoid Receptor Ligand Binding Domain Reveals a Novel Mode of Receptor Dimerization and Coactivator Recognition , 2002, Cell.

[75]  W. Pratt,et al.  Stepwise Assembly of a Glucocorticoid Receptor·hsp90 Heterocomplex Resolves Two Sequential ATP-dependent Events Involving First hsp70 and Then hsp90 in Opening of the Steroid Binding Pocket* , 2000, The Journal of Biological Chemistry.

[76]  David F. Smith,et al.  Hop as an Adaptor in the Heat Shock Protein 70 (Hsp70) and Hsp90 Chaperone Machinery* , 1998, The Journal of Biological Chemistry.

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

[78]  D. Toft,et al.  Hop Modulates hsp70/hsp90 Interactions in Protein Folding* , 1998, The Journal of Biological Chemistry.

[79]  S. Lindquist,et al.  In vivo functions of the Saccharomyces cerevisiae Hsp90 chaperone. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[80]  W. Pratt,et al.  Steroid receptor interactions with heat shock protein and immunophilin chaperones. , 1997, Endocrine reviews.

[81]  S. Bohen,et al.  Hsp90 Mutants Disrupt Glucocorticoid Receptor Ligand Binding and Destabilize Aporeceptor Complexes * , 1995, The Journal of Biological Chemistry.

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

[83]  K. Yamamoto,et al.  Isolation of Hsp90 mutants by screening for decreased steroid receptor function. , 1993, Proceedings of the National Academy of Sciences of the United States of America.

[84]  S. Lindquist,et al.  Reduced levels of hsp90 compromise steroid receptor action in vivo , 1990, Nature.

[85]  F. Boschelli,et al.  The Ydj1 molecular chaperone facilitates formation of active p60v-src in yeast. , 1996, Molecular biology of the cell.