Structure of Hsp90–Hsp70–Hop–GR reveals the Hsp90 client-loading mechanism

In the paper

“Finally, sequence alignments of Hsp90 and Hsp70 homologues and paralogues showed that interface I and II residues are generally conserved, suggesting a universal Hsp90–Hsp70 binding strategy across species23,24,27 and organelles28,29 (Supplementary Fig. 2a, b).”

What the sources say

• Partly supported

  The Results (page not available) align yeast Hsp90 interaction-region residues with their E. coli counterparts and report that three of five are identical or conserved; the other two are nonconserved. The Discussion (page not available) also links the yeast residues to an E. coli Hsp70-binding region. This supports a possible shared interaction region across those species, but not general conservation of both named interfaces among homologues and paralogues or a universal binding strategy. The organelle-specific citations in the sentence are separate from this source. (LLM, conf=0.86)

  cites [23] Kravats, A. N. et al. Functional and physical interaction between yeast Hsp90 and Hsp70. Proc. Natl Acad. Sci. USA 115, E2210–E2219 (2018). sourceExperimental — accuracy not yet measured
• Partly supported

  The Results section reports that direct Hsp90–Hsp70 interaction sites are conserved between bacteria and yeast. It does not report sequence alignments showing general conservation of interface I and II residues across homologues and paralogues, or establish a universal binding strategy across species and organelles. The Discussion describes conservation in higher eukaryotes as only possible; page numbers are unavailable in our copy. (LLM, conf=0.85)

  cites [24] Doyle, S. M. et al. Intermolecular interactions between Hsp90 and Hsp70. J. Mol. Biol. 431, 2729–2746 (2019). sourceExperimental — accuracy not yet measured
• ?Not checked — no full text of the sourcecites [27] Genest, O., Hoskins, J. R., Camberg, J. L., Doyle, S. M. & Wickner, S. Heat shock protein 90 from Escherichia coli collaborates with the DnaK chaperone system in client protein remodeling. Proc. Natl Acad. Sci. USA 108, 8206–8211 (2011). source
• Partly supported

  Section 3.4 reports Hsp90–Hsp70 cooperation in mitochondrial, eukaryotic cytosolic, and prokaryotic systems, and a direct interaction between TRAP1 and mitochondrial Hsp70. It does not report sequence alignments showing conservation of interface I and II residues or establish a universal binding strategy; the section instead describes a specific TRAP1–mortalin interaction. Page numbers were not available in the indexed passages. (LLM, conf=0.82)

  cites [28] Sung, N. et al. 2.4 A resolution crystal structure of human TRAP1NM, the Hsp90 paralog in the mitochondrial matrix. Acta Crystallogr. D 72, 904–911 (2016). sourceExperimental — accuracy not yet measured
• Partly supported

  The Discussion (page unavailable) reports that the ER pair BiP/Grp94 interacts through domains and mutation-sensitive sites similar to cytosolic Hsp70/Hsp90, supporting conservation between those compartments. It does not report the claimed interface I/II sequence alignments or establish a universal binding strategy across species and organelles. (LLM, conf=0.82)

  cites [29] Sun, M., Kotler, J. L. M., Liu, S. & Street, T. O. The endoplasmic reticulum (ER) chaperones BiP and Grp94 selectively associate when BiP is in the ADP conformation. J. Biol. Chem. 294, 6387–6396 (2019). sourceExperimental — accuracy not yet measured

In the paper

“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 release8.”

What the sources say

• Supported

  The Results section describes an inactive GR–Hsp90–Hsp70–HOP intermediate and places p23 later in the maturation pathway, after Hsp90 releases Hsp70’s inhibition. The Introduction identifies the p23-bound Hsp90 complex as ligand-binding competent, and the Abstract reports Hsp70-mediated inactivation followed by release of GR from Hsp90. Page numbers are not available in our copy. (LLM, conf=0.91)

  cites [8] Kirschke, E., Goswami, D., Southworth, D., Griffin, P. R. & Agard, D. A. Glucocorticoid receptor function regulated by coordinated action of the Hsp90 and Hsp70 chaperone cycles. Cell 157, 1685–1697 (2014). source

In the paper

“Notably, interface II defines the Hsp90ATP-like position and orientation of the Hsp90NTD with respect to the Hsp90MD, explaining the observation that Hsp70 accelerates Hsp90 ATPase activity25.”

What the sources say

• Partly supported

  The Results section (“ATP hydrolysis by Hsp90Ec and DnaK is synergistically stimulated”; page not provided) reports enhanced ATP hydrolysis by the two chaperones together in the presence of the client protein L2. It does not establish that Hsp70 specifically accelerates Hsp90’s ATPase, and the cited paper does not describe interface II positioning the Hsp90 N-terminal domain relative to its middle domain or propose that positioning as the explanation. (LLM, conf=0.86)

  cites [25] Genest, O., Hoskins, J. R., Kravats, A. N., Doyle, S. M. & Wickner, S. Hsp70 and Hsp90 of E. coli directly interact for collaboration in protein remodeling. J. Mol. Biol. 427, 3877–3889 (2015). sourceExperimental — accuracy not yet measured

In the paper

“The observation of the two-Hsp70 and one-Hsp70 loading complexes populated in our sample is consistent with a previous study20.”

What the sources say

• Supported

  The Results section (‘Antiparallel Dimers of Hsp70 Facilitate Client Transfer to Hsp90,’ Figure 4C) reports client-loading complexes containing either one or two Hsp70 molecules, with the two-Hsp70 complex predominant. The cited paper therefore supports the comparison to the citing authors’ sample; it does not establish what their sample contains. (LLM, conf=0.94)

  cites [20] Morgner, N. et al. Hsp70 forms antiparallel dimers stabilized by post-translational modifications to position clients for transfer to Hsp90. Cell Rep. 11, 759–769 (2015). source

In the paper

“This explains why mutations at interface I on either Hsp90 (G333, K414, K418, K419)21–23 or Hsp70 (R171, N174, D213)24 led to defective Hsp90-Hsp70 interaction, growth defects and impaired client maturation of GR, v-Src and luciferase (Fig. 1d, e, Extended Data Fig. 4c–h; see Supplementary Table 3 for equivalent yeast residue numbering).”

What the sources say

• Partly supported

  The 'Isolation of hsp82 ts mutants' section lists G313S among the Hsp90 mutations selected for temperature-sensitive growth, and the results report reduced GR and v-Src activity in the mutants. The copy does not establish that these mutations disrupt Hsp90–Hsp70 binding, or report impaired luciferase maturation; it also does not identify the cited interface-I residues by the numbering used in the claim. Pages were not provided for these passages. (LLM, conf=0.87)

  cites [21] Nathan, D. F. & Lindquist, S. Mutational analysis of Hsp90 function: interactions with a steroid receptor and a protein kinase. Mol. Cell. Biol. 15, 3917–3925 (1995). sourceExperimental — accuracy not yet measured
• ?Not checked — no full text of the sourcecites [22] Bohen, S. P. & Yamamoto, K. R. Isolation of Hsp90 mutants by screening for decreased steroid receptor function. Proc. Natl Acad. Sci. USA 90, 11424–11428 (1993). source
• Partly supported

  The Results report that mutations in the corresponding yeast Hsp90 region reduce interaction with Hsp70, cause growth defects at high temperature, impair GR activity, and prevent stimulation of luciferase reactivation. They do not establish impaired v-Src maturation. The Hsp70 residue mutations have a separate citation in the citing sentence and are not attributed to this paper. (LLM, conf=0.85)

  cites [23] Kravats, A. N. et al. Functional and physical interaction between yeast Hsp90 and Hsp70. Proc. Natl Acad. Sci. USA 115, E2210–E2219 (2018). sourceExperimental — accuracy not yet measured
• Partly supported

  The citation is attached to the Hsp70 residues, not the Hsp90 residues cited to other papers. In the Results section on Ssa1 substitution mutants (page unavailable), the source reports that yeast Hsp70 mutants R169H, N172D and E210R had reduced interaction with Hsp82 and were defective in reactivating heat-denatured luciferase. It does not establish growth defects or impaired GR and v-Src maturation, and the passage does not verify the citing sentence’s residue numbering. (LLM, conf=0.86)

  cites [24] Doyle, S. M. et al. Intermolecular interactions between Hsp90 and Hsp70. J. Mol. Biol. 431, 2729–2746 (2019). sourceExperimental — accuracy not yet measured

In the paper

“The structure of HopTPR2A-TRP2B closely matches the yeast crystal structure33 (Cα-RMSD of 1.47 Å to PDB ID 3UQ3, Supplementary Fig. 5a, c, d), including the conserved electrostatic network (Hop(Y354, R389, E385, K388)) that defines the unique interdomain angle (Supplementary Fig. 5h–j).”

What the sources say

• Supported

  The Materials and methods section identifies PDB 3UQ3 as the paper’s TPR2A–TPR2B crystal structure. The Results section describes a conserved linker interaction that fixes the domains’ orientation. Citation 33 identifies the yeast structure used for comparison; the numerical RMSD and Hop residue numbering are presented as the citing authors’ own comparison, not as results attributed to this source. (LLM, conf=0.90)

  cites [33] Schmid, A. B. et al. The architecture of functional modules in the Hsp90 co-chaperone Sti1/Hop. EMBO J. 31, 1506–1517 (2012). source

In the paper

“The cochaperone Hop is well-conserved in eukaryotes and facilitates GR maturation in vivo32 and in vitro8.”

What the sources say

• ?Not checked — no full text of the sourcecites [32] Sahasrabudhe, P., Rohrberg, J., Biebl, M. M., Rutz, D. A. & Buchner, J. The plasticity of the Hsp90 co-chaperone System. Mol. Cell 67, 947–961 (2017). source
• Supported

  Citation 8 is attached to the in vitro claim; the in vivo claim has a separate citation. In “Hsp90 Recovers GR Ligand Binding” (Results; page unavailable), the paper reports that HOP helps restore GR ligand binding in a reconstituted system, supporting its role in GR maturation in vitro. (LLM, conf=0.91)

  cites [8] Kirschke, E., Goswami, D., Southworth, D., Griffin, P. R. & Agard, D. A. Glucocorticoid receptor function regulated by coordinated action of the Hsp90 and Hsp70 chaperone cycles. Cell 157, 1685–1697 (2014). source

In the paper

“Notably, these three domains are necessary and sufficient for full GR activation33.”

What the sources say

• Partly supported

  The Results (Figure 5A; page not provided) show that the three-domain TPR2A–TPR2B–DP2 fragment supports GR activation, and that removing DP2 abolishes Sti1 function in this assay. But the fragment achieved 77% of wild-type GR activation, not full activation, so the sufficiency claim is overstated. (LLM, conf=0.91)

  cites [33] Schmid, A. B. et al. The architecture of functional modules in the Hsp90 co-chaperone Sti1/Hop. EMBO J. 31, 1506–1517 (2012). sourceExperimental — accuracy not yet measured

In the paper

“The canonical nucleotide exchange factor (NEF)-binding sites10,14 on Hsp70NBD-IIB remain available, explaining how the NEF Bag-1 can accelerate GR maturation30 (Supplementary Fig. 4a, b) and also how the NEF Hsp110 can be involved in GR maturation31 (Supplementary Fig. 4c, d).”

What the sources say

• Supported

  Citation 30 is attached to the claim that Bag-1 can accelerate GR maturation. In “Bag-1 Functions Synergistically with Hsp90 to Facilitate GRLBD Reactivation” (page unavailable), the paper reports that Bag-1 accelerates GRLBD reactivation within the Hsp90 pathway. The binding-site and Hsp110 statements have other citations and are not attributed to this source. (LLM, conf=0.92)

  cites [30] Kirschke, E., Roe-Zurz, Z., Noddings, C. & Agard, D. The interplay between Bag-1, Hsp70, and Hsp90 reveals that inhibiting Hsp70 rebinding is essential for glucocorticoid receptor activity. Preprint at https://doi.org/10.1101/2020.05.03.075523 (2020). source
• Supported

  Citation 31 supports the clause that Hsp110 can participate in GR maturation. In Results (‘The Role of Sse1 in Hsp90 Client Protein Fate Determination’), the paper reports that the Hsp110 protein Sse2 completely restored GR activation in cells lacking Sse1. The claim about available NEF-binding sites is not attributed to this citation. (LLM, conf=0.92)

  cites [31] Mandal, A. K. et al. Hsp110 chaperones control client fate determination in the hsp70-Hsp90 chaperone system. Mol. Biol. Cell 21, 1439–1448 (2010). source

In the paper

“Hop is thought to bring Hsp90 and Hsp70 together using its three tetratricopeptide repeat (TPR) domains that bind the EEVD C termini on both Hsp90 and Hsp7033 (Extended Data Fig. 1a).”

What the sources say

• Supported

  The Introduction says Sti1/Hop’s TPR domains bind the C-terminal stretches of Hsp90 and Hsp70, and the Abstract identifies its three TPR domains and reports simultaneous binding of the two chaperones. The Results identify MEEVD as the Hsp90 C-terminal peptide and show binding of the Hsp70 C-terminal peptide PTVEEVD. (LLM, conf=0.94)

  cites [33] Schmid, A. B. et al. The architecture of functional modules in the Hsp90 co-chaperone Sti1/Hop. EMBO J. 31, 1506–1517 (2012). source

In the paper

“Not only does it interact with GR and HopDP2 (Fig. 2d), but it also interacts with another cochaperone, Aha142.”

What the sources say

• Partly supported

  The section ‘Aha1 stabilizes AMPPNP-bound closed states of Hsp90’ (page unavailable) shows that Aha1 interacts with Hsp90, supporting the part of the sentence to which this citation is attached. It identifies Hsp90 residues W296 and F328 at the Aha1 interface, however, not W320; our copy therefore does not establish the claimed W320–Aha1 interaction. The GR and HopDP2 interactions are attributed to the citing paper’s Fig. 2d, not to this source. (LLM, conf=0.86)

  cites [42] Liu, Y. et al. Cryo-EM structures reveal a multistep mechanism of Hsp90 activation by co-chaperone Aha1. Preprint at https://doi.org/10.1101/2020.06.30.180695 (2020). sourceExperimental — accuracy not yet measured

In the paper

“Mutations of this residue decrease yeast viability (Hop(L508D)) (Extended Data Fig. 8g), and completely abrogate the function of GR (Hop(L508A); ref. 33) and v-Src (Hop(L508D)) (Extended Data Fig. 8h) in vivo.”

What the sources say

• Partly supported

  The Results (page not supplied) report that yeast Sti1 L553A produced only background levels of GR activity in vivo, supporting the claimed loss of GR function. The cited passage does not identify this mutation as Hop L508A or establish that the two residues correspond. The viability and v-Src findings are attributed to the citing paper’s Extended Data figures, not to this source. (LLM, conf=0.83)

  cites [33] Schmid, A. B. et al. The architecture of functional modules in the Hsp90 co-chaperone Sti1/Hop. EMBO J. 31, 1506–1517 (2012). sourceExperimental — accuracy not yet measured

In the paper

“Our HopDP2 structure agrees well with the yeast nuclear magnetic resonance (NMR) structure33 (Cα-RMSD of 1.13 Å to PDB 1D 2LLW) (Supplementary Fig. 5b), adopting a hand-like α-helical structure, with many of its core hydrophobic sidechains exposed in the ‘palm’ of the ‘hand’ (Fig. 3d, Extended Data Fig. 8a).”

What the sources say

• Supported

  The citation identifies the yeast NMR structure used for comparison, rather than attributing the citing authors’ RMSD calculation or description of their own HopDP2 structure to this paper. In Results (‘The DP domains represent a novel α-helical fold’; page unavailable), the cited paper reports determining the Sti1 DP2 fold by NMR and describes its five-helix structure. (LLM, conf=0.91)

  cites [33] Schmid, A. B. et al. The architecture of functional modules in the Hsp90 co-chaperone Sti1/Hop. EMBO J. 31, 1506–1517 (2012). source

In the paper

“Re-examination of previous Hsp70-GR hydrogen deuterium exchange mass spectrometry (HDX-MS) data8 reveals that only GRpre-Helix1 becomes protected upon Hsp70 binding (Extended Data Fig. 9a,b) and GRpre-Helix1 also contains high-scoring predicted Hsp70-binding sites (GR519–526) (Extended Data Fig. 9a).”

What the sources say

• Supported

  Citation 8 identifies the previous Hsp70–GR HDX-MS data, not the citing authors’ re-examination or the predicted binding sites, which are attributed to their Extended Data figures. The source paper’s Results section, “Hsp70 Inhibits GR Ligand Binding by Partially Unfolding GR” (page unavailable), reports HDX-MS measurements of GR in the presence of Hsp70 and Hsp40. (LLM, conf=0.92)

  cites [8] Kirschke, E., Goswami, D., Southworth, D., Griffin, P. R. & Agard, D. A. Glucocorticoid receptor function regulated by coordinated action of the Hsp90 and Hsp70 chaperone cycles. Cell 157, 1685–1697 (2014). source

In the paper

“Of note, in the loading complex, Hsp70NBD-IIAs deviate from the crystal structure (Supplementary Fig. 3a, b) and Hsp90MD interacts with Hsp70 (R171, N174, T177) in the ATP catalytic motif (Fig. 1e, Supplementary Fig. 3c), suggesting how Hsp90 enhances the nucleotide exchange on Hsp70 during the GR-chaperoning cycle30.”

What the sources say

• Partly supported

  Split into 2 atoms — 1 confirmed, 0 partially supported, 0 disputed, 0 misattributed, 1 unverifiable. Expand the claim to see per-atom verdicts.

  cites [30] Kirschke, E., Roe-Zurz, Z., Noddings, C. & Agard, D. The interplay between Bag-1, Hsp70, and Hsp90 reveals that inhibiting Hsp70 rebinding is essential for glucocorticoid receptor activity. Preprint at https://doi.org/10.1101/2020.05.03.075523 (2020). sourceExperimental — accuracy not yet measured

In the paper

“Supporting its broad functional importance, numerous studies have reported deleterious effects of Hsp90 W320 mutations on GR activation40,43 and yeast growth26.”

What the sources say

• Partly supported

  This citation is attached to the yeast-growth portion of the sentence, not the GR-activation portion. The Results report a severe growth defect for the W300A yeast strain, including failure to grow at 37°C, but our copy does not identify that mutation as W320 or establish a numbering equivalence. (LLM, conf=0.84)

  cites [40] Hawle, P. et al. The middle domain of Hsp90 acts as a discriminator between different types of client proteins. Mol. Cell. Biol. 26, 8385–8395 (2006). sourceExperimental — accuracy not yet measured
• Partly supported

  The Results section, “Tryptophan 300 is essential for viability and GR maturation” (page unavailable), reports a severe yeast growth defect for Hsp90 W300A and nonviability for W300E/K. It also reports reduced GR activation for W300A. The cited paper identifies these mutations as W300 in yeast, not W320, and does not establish that its findings apply to W320 mutations. (LLM, conf=0.85)

  cites [43] Rutz, D. A. et al. A switch point in the molecular chaperone Hsp90 responding to client interaction. Nat. Commun. 9, 1472 (2018). sourceExperimental — accuracy not yet measured
• ?Could not verify

  The Results report that substitutions at Hsp90 W300 can cause severe yeast-growth defects, but our copy does not identify W300 as W320 or report growth effects for W320 mutations. The GR-activation claim is attached to other citations in the citing sentence; this citation is attached to yeast growth. Searches for W320 and GR activation found no decisive passage. (LLM, conf=0.72)

  cites [26] Flynn, J. M. et al. Comprehensive fitness maps of Hsp90 show widespread environmental dependence. eLife 9, e58310 (2020). sourceExperimental — accuracy not yet measured

In the paper

“Model overfitting was monitored and contained using the half-map approach as previously described64,65, in which one-half map from RELION Refine3d was used for density-guided refinement, whereas the other half map was used for validation.”

What the sources say

• Supported

  The Introduction describes splitting particle images into two sets, using one reconstructed map for model refinement and the other for cross-validation. The Results confirm that the testing map was held aside until validation, and assess overfitting by comparing model fit to the training and testing maps. The reference to RELION Refine3d describes the citing paper’s implementation, not a method attributed to this source. (LLM, conf=0.93)

  cites [64] DiMaio, F., Zhang, J., Chiu, W. & Baker, D. Cryo-EM model validation using independent map reconstructions. Protein Sci. 22, 865–868 (2013). source
• Supported

  The Materials and methods section ('Preparing maps for refinement') says one split map was chosen for refinement and the other used for validation. The Figure 1 description also says refinement used the training half-map while models were selected using agreement with the independently constructed validation half-map, helping avoid overfitting. (LLM, conf=0.94)

  cites [65] Wang, R. Y. et al. Automated structure refinement of macromolecular assemblies from cryo-EM maps using Rosetta. Elife 5, e17219 (2016). source

In the paper

“As previously shown23, Δsti1hsc82hsp82 cells expressing hsc82-G309S are viable in the presence of STI1 but inviable in Δsti1 cells.”

What the sources say

• Supported

  The Results report that cells expressing Hsc82-G309S as their sole Hsp90 source grew at 30 °C when Sti1 was present, whereas sti1Δ cells expressing the mutant could not grow at 30 °C. Materials and Methods identifies the corresponding hsc82/hsp82 double-deletion strains used for these tests. The viability comparison is supported under the tested temperature conditions. (LLM, conf=0.93)

  cites [23] Kravats, A. N. et al. Functional and physical interaction between yeast Hsp90 and Hsp70. Proc. Natl Acad. Sci. USA 115, E2210–E2219 (2018). source

In the paper

“The resulting completed models of HopTPR2A and HopTPR2B showed a high resemblance to their structures determined by NMR63 individually (Supplementary Fig. 5c, d).”

What the sources say

• Partly supported

  The Results and discussion section (‘NMR solution structure of the Hop TPR2A domain’; page unavailable) reports an NMR structure for Hop TPR2A. It does not report an NMR structure for Hop TPR2B, so it supports only one of the two reference structures invoked. The resemblance of the citing authors’ completed models is their own comparison, not a finding attributed to this source. (LLM, conf=0.84)

  cites [63] Darby, J. F. et al. Solution structure of the Hop TPR2A domain and investigation of target druggability by NMR, biochemical and in silico approaches. Sci. Rep. 10, 16000 (2020). sourceExperimental — accuracy not yet measured

In the paper

“Similar to the interface I mutations G333S and K418E in Hsp90 (G309S and K394E in yeast Hsc82), an Hsp90 mutation at R60 (yHsc82(R46G)) led to reduced Hsp70 interaction, inviability at 37 °C and reduced v-Src activity (Extended Data Fig. 4e–h).”

In the paper

“Two major interfaces (Fig. 1d) are formed in both of the nearly identical Hsp70NBD–Hsp90 protomer interactions (root mean square deviation (RMSD) of 0.96 Å) (Supplementary Fig. 1a–c).”

In the paper

“GR is partially unfolded and recognized through an extended binding pocket composed of Hsp90, Hsp70 and Hop, revealing the mechanism of GR loading and inactivation.”

In the paper

“As expected from Hsp90(D93N), both Hsp90NTD ATP-binding pockets are empty with their lids open.”

In the paper

“Interface I is tightly packed (approximately 479 Å2 of buried surface area; BSA), and is stabilized by numerous polar interactions (Fig. 1d, e).”

In the paper

“Hop interacts with all components of the complex, including GR, and poises Hsp90 for subsequent ATP hydrolysis.”

In the paper

“Hop intimately interacts with each Hsp90 protomer, the two Hsp70s and, notably, a portion of GR.”

In the paper

“The two Hsp70NBDs clearly have ADP bound and are similar to the ADP-bound Hsp70NBD crystal structure (Cα-RMSD of 0.50 Å (Hsp70C) and 0.53 Å (Hsp70S) to PDB ID 3AY9) (Fig. 1d, Supplementary Fig. 3a, b).”

In the paper

“Focused maps revealed that HopTPR2A and HopTPR2B are bound to the EEVD termini of Hsp90 and Hsp70, respectively (Extended Data Fig. 6d–f, Supplementary Fig. 6b–f, k, l).”

In the paper

“In addition, a residue in the HopDP2 hydrophobic palm (Hop L508; L553 in yeast, Sti1) that directly interacts with GR (Figs. 2d, 3d, Extended Data Fig. 8e) is functionally important.”

In the paper

“Y296A–A328H mutations of Hop (Y332–T364 in yeast, Sti1) at the Hsp70SNBD-IIA–HopTPR2A interface (Extended Data Fig. 6a–c, g) resulted in yeast growth defects (Extended Data Fig. 6i) and did not promote v-Src maturation (Extended Data Fig. 6j).”

In the paper

“Hop wraps around much of the loading complex, with extensive interactions made by HopTPR2A and HopDP2, demonstrating a far more integral role than was anticipated (Fig. 2a, c).”

In the paper

“On the other side, a GR helix is cradled in the HopDP2 hydrophobic palm and the rest of GR becomes a strand embedded in the Hsp70CSBD-β substrate-binding pocket (Fig. 3a–d, Extended Data Figs. 8b, 9d, Supplementary Figs. 8a, b, 9d–f).”

In the paper

“Nonetheless, the Hsp90MDs from each protomer and the Hsp90Bamphi-α clearly contact GR (Extended Data Fig. 7b–d, f, g).”

In the paper

“Hsp70SNBD-IIA interacts with Hop and Hsp90 simultaneously, thereby rigidly positioning Hop with respect to Hsp90.”

In the paper

“The rest of GR remains globular and is only loosely associated with the distal surface of Hsp90.”

In the paper

“In support of our structure, GR and Hop become photo-cross-linked (Extended Data Fig. 8c, d).”

In the paper

“Viability of sti1hsc82hsp82 cells expressing hsc82-G309S is restored by the presence of a plasmid expressing wild-type STI1 but not mutant sti1.”

In the paper

“The loading complex presents conformational heterogeneity at all regions of the complex, in particular at the HopTPR2A-TPR2B (Supplementary Fig. 5f–h).”

In the paper

“All strains exhibited similar growth in the presence of glucose.”

In the paper

“Note that the N-terminal MBP (maltose-binding protein) tag of the GRLBD construct was ruled out to be the density connected to the GRpre-Helix 1 (Supplementary Fig. 8c).”

In the paper

“The focused maps showed much better atomic details than the global reconstruction at all regions, and hence were used for model building and refinement.”

In the paper

“Among eight classes, one class (around 8 Å resolution) showed recognizable shapes of the protein components, although the class is markedly different from the initial model (Extended Data Fig. 1d).”

In the paper

“Of note, in Hsp70ATP this cleft binds the Hsp70 interdomain linker and also contributes to binding to the Hsp40 J-domain14 (Extended Data Fig. 4b). Hence, the cleft is only available for Hsp90 in Hsp70ADP.”

What the sources say

• ?Not checked — no full text of the sourcecites [14] Mayer, M. P. & Gierasch, L. M. Recent advances in the structural and mechanistic aspects of Hsp70 molecular chaperones. J. Biol. Chem. 294, 2085–2097 (2019). source

In the paper

“Our structural insights reveal the molecular mechanism of GR inactivation in the chaperone cycle (Fig. 1a), allowing us to propose the following pathway for loading complex formation (Fig. 4b):”

In the paper

“Our structural data also enable us to propose how the loading complex progresses to the maturation complex–a process requiring Hsp90 ATP hydrolysis and release of Hop and both Hsp70s.”

In the paper

“This leaves GR engaged with HopDP2 and the Hsp90 lumen, minimizing reformation of an Hsp70–GR complex or premature release.”

In the paper

“Despite extensive 3D classifications, the main body of GR remained at low resolution.”

In the paper

“For Hsp70 to re-enter its ATP cycle, it must first leave Hsp90, thus nucleotide exchange on Hsp70 is likely to time its dissociation.”

In the paper

“These data indicate that client binding to HopDP2 is crucial for both general cellular function and protein maturation across different Hsp90 client systems.”

In the paper

“The one-Hsp70 loading complex (Extended Data Fig. 3d, e) suggests that the process is asymmetric and sequential, with the loss of Hsp70C occurring first.”

In the paper

“Not only is the loading complex lumen spacious enough to bind a strand (as shown here) or intact helix (Extended Data Fig. 3f), but the flexible positioning of the Hsp70CSBD and the dynamic, adaptable conformation of the Hsp90amphi-α allow even broader flexibility for client recognition.”

In the paper

“This suggests that Hop may first prepare Hsp90 for Hsp70 and client interaction, and subsequently rearrange after Hsp70SNBD binding (Supplementary Fig. 7d, e).”

In the paper

“Hsp70C captures the flexible GRpre-Helix 1, causing the next dynamic helix-strand motif to detach, thereby destabilizing the GR ligand-binding pocket (Extended Data Fig. 9b, c).”

In the paper

“Thus, GR is partially unfolded and threaded through the Hsp90 lumen.”

In the paper

“Together, this indicates that perturbations to GRHelix1 by Hsp70 or the loading complex leads to loss of GR ligand binding, explaining the inactivation of GR during the chaperone cycle.”

In the paper

“In addition, both positions were able to cross-link with Hop (Extended Data Fig. 10b, c), indicating that it is Hsp70C that the GRpre-Helix1 cross-linked with, rather than Hsp70S.”

In the paper

“In the resultant loading complex, GR is further unfolded through engagement of the LXXLL motif of GRHelix1 with HopDP2 and the GRpost-Helix1 strand with the Hsp90 lumen (Fig. 3b–d, Extended Data Fig. 8e), suppressing any possible ligand binding.”

In the paper

“Consistent with our model, a previous optical-tweezer study38 showed that GRHelix1 is readily detached, correlating with ligand binding loss.”

What the sources say

• ?Not checked — no full text of the sourcecites [38] Suren, T. et al. Single-molecule force spectroscopy reveals folding steps associated with hormone binding and activation of the glucocorticoid receptor. Proc. Natl Acad. Sci. USA 115, 11688–11693 (2018). source

In the paper

“Hsp90 repurposes one side of its client-binding sites to bind HopDP2, which in turn augments the Hsp90 lumenal client-binding site, facilitating client-loading from Hsp70 (Figs. 3b, 4a).”

In the paper

“Note that the dynamic range of the fitted atomic B-factors (Extended Data Fig. 2d, e) accurately represents the distribution of local-resolution estimates in the global reconstruction shown in Extended Data Fig. 2a.”

In the paper

“To ensure that pBpa-Hop (Extended Data Fig. 8a, b, d) and pBpa-GR (Extended Data Fig. 10) cross-link the bound segment in the context of the loading complex, cross-linking reactions were performed immediately after the complex was fractionated from SEC (see the previous section).”