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

May combine two claims.

Sequence alignments show that interface I and II residues are generally conserved among Hsp90 and Hsp70 homologues and paralogues, suggesting a universal Hsp90-Hsp70 binding strategy across species and organelles.

“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).”

• 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). sourcePartly 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)

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

  Experimental — accuracy not yet measured
• cites [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). sourceNot checked — no full text of the source
• 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). sourcePartly 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)

  Experimental — accuracy not yet measured
• 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). sourcePartly 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)

  Experimental — accuracy not yet measured

May combine two claims.

Interface II defines the Hsp90ATP-like position and orientation of the Hsp90NTD relative to the Hsp90MD, explaining why Hsp70 accelerates Hsp90 ATPase activity.

“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.”

• 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). sourcePartly 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)

  Experimental — accuracy not yet measured

May combine two claims.

The sample contains both two-Hsp70 and one-Hsp70 loading complexes, consistent with a previous study.

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

• 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). sourceSupported

• AssumptionRests on an assumption: The reconstituted loading complex containing Hsp90(D93N) and excess ADP represents the native client-loading intermediate of the GR chaperone cycle.
• AssumptionRests on an assumption: The structural features observed in the cryo-EM map reflect the solution conformation of the complex and are not artifacts of glutaraldehyde stabilization or cryo-EM sample preparation.

May combine two claims.

Mutations at interface I on Hsp90 (G333, K414, K418, K419) or Hsp70 (R171, N174, D213) cause defective Hsp90-Hsp70 interaction, growth defects, and impaired maturation of GR, v-Src and luciferase.

“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).”

• 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). sourcePartly 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)

  Experimental — accuracy not yet measured
• cites [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). sourceNot checked — no full text of the source
• 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). sourcePartly 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)

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

  Experimental — accuracy not yet measured

May combine two claims.

The HopTPR2A-TRP2B structure closely matches the yeast crystal structure, with Cα-RMSD 1.47 Å to PDB 3UQ3, including a conserved electrostatic network (Hop Y354, R389, E385, K388) that defines the unique interdomain angle.

“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).”

• 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). sourceSupported

• AssumptionRests on an assumption: The reconstituted loading complex containing Hsp90(D93N) and excess ADP represents the native client-loading intermediate of the GR chaperone cycle.
• AssumptionRests on an assumption: The structural features observed in the cryo-EM map reflect the solution conformation of the complex and are not artifacts of glutaraldehyde stabilization or cryo-EM sample preparation.

May combine two claims.

The three C-terminal Hop domains are necessary and sufficient for full GR activation.

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

• 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). sourcePartly 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)

  Experimental — accuracy not yet measured

May combine two claims.

The canonical NEF-binding sites on Hsp70NBD-IIB remain available in the loading complex, explaining how the NEF Bag-1 can accelerate GR maturation and how the NEF Hsp110 can participate in GR maturation.

“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).”

• 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). sourceSupported
• 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). sourceSupported

Probably not a substantial claim: may not pass S3 (load-bearing).

Hop is thought to bring Hsp90 and Hsp70 together through its three TPR domains, which bind the EEVD C termini of both Hsp90 and Hsp70.

“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).”

• 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). sourceSupported

May combine two claims.

Hsp90 W320 interacts with GR, HopDP2, and another cochaperone, Aha1.

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

• 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). sourcePartly 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)

  Experimental — accuracy not yet measured

• AssumptionRests on an assumption: The reconstituted loading complex containing Hsp90(D93N) and excess ADP represents the native client-loading intermediate of the GR chaperone cycle.
• AssumptionRests on an assumption: The structural features observed in the cryo-EM map reflect the solution conformation of the complex and are not artifacts of glutaraldehyde stabilization or cryo-EM sample preparation.

May combine two claims.

Mutations of Hop L508 decrease yeast viability (L508D) and completely abrogate GR function (L508A) and v-Src function (L508D) in vivo.

“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.”

• 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). sourcePartly 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)

  Experimental — accuracy not yet measured

May combine two claims.

The HopDP2 structure agrees well with the yeast NMR structure, with Cα-RMSD 1.13 Å to PDB 2LLW, adopting a hand-like α-helical structure with many core hydrophobic sidechains exposed in the palm.

“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).”

• 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). sourceSupported

• AssumptionRests on an assumption: The reconstituted loading complex containing Hsp90(D93N) and excess ADP represents the native client-loading intermediate of the GR chaperone cycle.
• AssumptionRests on an assumption: The structural features observed in the cryo-EM map reflect the solution conformation of the complex and are not artifacts of glutaraldehyde stabilization or cryo-EM sample preparation.

May combine two claims.

In the loading complex, Hsp70NBD-IIAs deviate from the crystal structure and Hsp90MD interacts with Hsp70 residues R171, N174 and T177 in the ATP catalytic motif, suggesting how Hsp90 enhances nucleotide exchange on Hsp70 during the GR-chaperoning cycle.

“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.”

• 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). sourcePartly supported

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

  Experimental — accuracy not yet measured

Probably not a substantial claim: may not pass S3 (load-bearing).

Numerous studies report deleterious effects of Hsp90 W320 mutations on GR activation and yeast growth.

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

• 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). sourcePartly 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)

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

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

  Experimental — accuracy not yet measured

Probably not a substantial claim: may not pass S3 (load-bearing).

Overfitting of the model was monitored and controlled with a half-map approach: one half-map was used for density-guided refinement and the other for validation.

“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.”

• cites [64] DiMaio, F., Zhang, J., Chiu, W. & Baker, D. Cryo-EM model validation using independent map reconstructions. Protein Sci. 22, 865–868 (2013). sourceSupported
• cites [65] Wang, R. Y. et al. Automated structure refinement of macromolecular assemblies from cryo-EM maps using Rosetta. Elife 5, e17219 (2016). sourceSupported

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

Previous work showed that Δsti1hsc82hsp82 cells expressing hsc82-G309S are viable when STI1 is present but not viable in Δsti1 cells.

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

• 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). sourceSupported

Probably not a substantial claim: may not pass S3 (load-bearing).

The completed HopTPR2A and HopTPR2B models closely resembled their individual NMR structures.

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

• 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). sourcePartly 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)

  Experimental — accuracy not yet measured

May combine two claims.

An Hsp90 mutation at R60 (yHsc82(R46G)) reduces Hsp70 interaction, causes inviability at 37 °C, and reduces v-Src activity.

“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).”

May combine two claims.

Two major interfaces form in both nearly identical Hsp70NBD-Hsp90 protomer interactions, with an RMSD of 0.96 Å.

“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).”

• AssumptionRests on an assumption: The reconstituted loading complex containing Hsp90(D93N) and excess ADP represents the native client-loading intermediate of the GR chaperone cycle.
• AssumptionRests on an assumption: The structural features observed in the cryo-EM map reflect the solution conformation of the complex and are not artifacts of glutaraldehyde stabilization or cryo-EM sample preparation.

May combine two claims.

GR is partially unfolded and is recognized by an extended binding pocket made of Hsp90, Hsp70 and Hop.

“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.”

• AssumptionRests on an assumption: The reconstituted loading complex containing Hsp90(D93N) and excess ADP represents the native client-loading intermediate of the GR chaperone cycle.
• AssumptionRests on an assumption: The structural features observed in the cryo-EM map reflect the solution conformation of the complex and are not artifacts of glutaraldehyde stabilization or cryo-EM sample preparation.

May combine two claims.

Both Hsp90 NTD ATP-binding pockets in the loading complex are empty and have open lids.

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

• AssumptionRests on an assumption: The reconstituted loading complex containing Hsp90(D93N) and excess ADP represents the native client-loading intermediate of the GR chaperone cycle.
• AssumptionRests on an assumption: The structural features observed in the cryo-EM map reflect the solution conformation of the complex and are not artifacts of glutaraldehyde stabilization or cryo-EM sample preparation.

May combine two claims.

Interface I is tightly packed, with about 479 Å2 of buried surface area, and is stabilized by many polar interactions.

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

• AssumptionRests on an assumption: The reconstituted loading complex containing Hsp90(D93N) and excess ADP represents the native client-loading intermediate of the GR chaperone cycle.
• AssumptionRests on an assumption: The structural features observed in the cryo-EM map reflect the solution conformation of the complex and are not artifacts of glutaraldehyde stabilization or cryo-EM sample preparation.

May combine two claims.

Hop contacts all components of the GR-loading complex, including GR, and positions Hsp90 for later ATP hydrolysis.

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

• AssumptionRests on an assumption: The reconstituted loading complex containing Hsp90(D93N) and excess ADP represents the native client-loading intermediate of the GR chaperone cycle.
• AssumptionRests on an assumption: The structural features observed in the cryo-EM map reflect the solution conformation of the complex and are not artifacts of glutaraldehyde stabilization or cryo-EM sample preparation.

May combine two claims.

Hop interacts intimately with each Hsp90 protomer, both Hsp70 molecules, and part of GR.

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

• AssumptionRests on an assumption: The reconstituted loading complex containing Hsp90(D93N) and excess ADP represents the native client-loading intermediate of the GR chaperone cycle.
• AssumptionRests on an assumption: The structural features observed in the cryo-EM map reflect the solution conformation of the complex and are not artifacts of glutaraldehyde stabilization or cryo-EM sample preparation.

May combine two claims.

The two Hsp70 NBDs in the loading complex have ADP bound and resemble the ADP-bound Hsp70NBD crystal structure, with Cα-RMSD values of 0.50 Å for Hsp70C and 0.53 Å for Hsp70S to PDB 3AY9.

“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).”

• AssumptionRests on an assumption: The reconstituted loading complex containing Hsp90(D93N) and excess ADP represents the native client-loading intermediate of the GR chaperone cycle.
• AssumptionRests on an assumption: The structural features observed in the cryo-EM map reflect the solution conformation of the complex and are not artifacts of glutaraldehyde stabilization or cryo-EM sample preparation.

May combine two claims.

Focused maps show HopTPR2A and HopTPR2B bound to the EEVD termini of Hsp90 and Hsp70, respectively.

“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).”

• AssumptionRests on an assumption: The reconstituted loading complex containing Hsp90(D93N) and excess ADP represents the native client-loading intermediate of the GR chaperone cycle.
• AssumptionRests on an assumption: The structural features observed in the cryo-EM map reflect the solution conformation of the complex and are not artifacts of glutaraldehyde stabilization or cryo-EM sample preparation.

May combine two claims.

A residue in the HopDP2 hydrophobic palm (Hop L508; L553 in yeast, Sti1) directly interacts with GR and is functionally important.

“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.”

• AssumptionRests on an assumption: The reconstituted loading complex containing Hsp90(D93N) and excess ADP represents the native client-loading intermediate of the GR chaperone cycle.
• AssumptionRests on an assumption: The structural features observed in the cryo-EM map reflect the solution conformation of the complex and are not artifacts of glutaraldehyde stabilization or cryo-EM sample preparation.

May combine two claims.

Hop mutations Y296A-A328H at the Hsp70SNBD-IIA-HopTPR2A interface cause yeast growth defects and do not promote v-Src maturation.

“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).”

May combine two claims.

Hop wraps around much of the loading complex, with extensive interactions made by HopTPR2A and HopDP2, showing a more integral role than anticipated.

“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).”

• AssumptionRests on an assumption: The reconstituted loading complex containing Hsp90(D93N) and excess ADP represents the native client-loading intermediate of the GR chaperone cycle.
• AssumptionRests on an assumption: The structural features observed in the cryo-EM map reflect the solution conformation of the complex and are not artifacts of glutaraldehyde stabilization or cryo-EM sample preparation.

May combine two claims.

Re-examination of previous Hsp70-GR HDX-MS data shows that only GRpre-Helix1 becomes protected upon Hsp70 binding, and GRpre-Helix1 contains high-scoring predicted Hsp70-binding sites (GR519-526).

“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).”

• 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). sourceNot checked — no full text of the source

May combine two claims.

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.

“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).”

• AssumptionRests on an assumption: The reconstituted loading complex containing Hsp90(D93N) and excess ADP represents the native client-loading intermediate of the GR chaperone cycle.
• AssumptionRests on an assumption: The structural features observed in the cryo-EM map reflect the solution conformation of the complex and are not artifacts of glutaraldehyde stabilization or cryo-EM sample preparation.

May combine two claims.

The Hsp90MDs from each protomer and Hsp90Bamphi-α clearly contact GR.

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

• AssumptionRests on an assumption: The reconstituted loading complex containing Hsp90(D93N) and excess ADP represents the native client-loading intermediate of the GR chaperone cycle.
• AssumptionRests on an assumption: The structural features observed in the cryo-EM map reflect the solution conformation of the complex and are not artifacts of glutaraldehyde stabilization or cryo-EM sample preparation.

May combine two claims.

Hsp70SNBD-IIA interacts with Hop and Hsp90 simultaneously, rigidly positioning Hop relative to Hsp90.

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

• AssumptionRests on an assumption: The reconstituted loading complex containing Hsp90(D93N) and excess ADP represents the native client-loading intermediate of the GR chaperone cycle.
• AssumptionRests on an assumption: The structural features observed in the cryo-EM map reflect the solution conformation of the complex and are not artifacts of glutaraldehyde stabilization or cryo-EM sample preparation.

May combine two claims.

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

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

• AssumptionRests on an assumption: The reconstituted loading complex containing Hsp90(D93N) and excess ADP represents the native client-loading intermediate of the GR chaperone cycle.
• AssumptionRests on an assumption: The structural features observed in the cryo-EM map reflect the solution conformation of the complex and are not artifacts of glutaraldehyde stabilization or cryo-EM sample preparation.

May combine two claims. The quote only partly states this claim.

When Hop Q512 is substituted with p-benzoyl-phenylalanine, GR and Hop become photo-cross-linked, supporting the structural interaction.

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

May combine two claims.

In sti1hsc82hsp82 cells expressing hsc82-G309S, viability is restored by a plasmid carrying wild-type STI1 but not by mutant sti1.

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

May combine two claims.

The loading complex is conformationally heterogeneous in all regions, especially at HopTPR2A-TPR2B.

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

Probably not a substantial claim: may not pass S3 (load-bearing).

All strains grew similarly on glucose.

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

Probably not a substantial claim: may not pass S3 (load-bearing).

The N-terminal MBP tag of the GRLBD construct was excluded as the density connected to GRpre-Helix 1.

“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).”

Classified as probably not a substantial claim. May combine two claims.

The focused maps gave better atomic detail than the global reconstruction in all regions, so they were used for model building and refinement.

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

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

In 3D classification, one of eight classes, at about 8 Å resolution, displayed recognizable protein shapes, although it differed markedly from the initial model.

“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).”

May combine two claims.

The GR chaperone cycle proceeds through Hsp70-mediated inactivation, formation of an inactive GR-Hsp90-Hsp70-Hop loading complex, conversion to an active GR-Hsp90-p23 maturation complex, and release of GR.

“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.”

• 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). sourceNot checked — no full text of the source

May combine two claims.

In Hsp70ATP, the cleft that binds Hsp90 at interface I is occupied by the Hsp70 interdomain linker and contributes to Hsp40 J-domain binding, so the cleft is available for Hsp90 only in Hsp70ADP.

“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.”

• cites [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). sourceNot checked — no full text of the source

May combine two claims.

Hop is well conserved in eukaryotes and facilitates GR maturation in vivo and in vitro.

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

• cites [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). sourceNot checked — no full text of the source
• 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). sourceNot checked — no full text of the source

May combine two claims.

The structural insights reveal the molecular mechanism of GR inactivation in the chaperone cycle and allow proposing a pathway for loading complex formation.

“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):”

May combine two claims.

The change from the loading complex to the maturation complex needs Hsp90 to hydrolyse ATP and to release Hop and both Hsp70 molecules.

“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.”

May combine two claims.

After Hsp70C leaves, GR stays bound to HopDP2 and the Hsp90 lumen, which limits re-formation of an Hsp70-GR complex and premature release.

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

Probably not a substantial claim: may not pass S3 (load-bearing).

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

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

May combine two claims.

For Hsp70 to re-enter its ATP cycle, it must first leave Hsp90, so nucleotide exchange on Hsp70 likely times its dissociation.

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

May combine two claims.

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

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

May combine two claims.

The transition occurs asymmetrically and in sequence, with Hsp70C leaving first.

“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.”

May combine two claims.

The loading complex lumen is spacious enough to bind a strand or an intact helix, and the flexible positioning of Hsp70CSBD and the dynamic conformation of Hsp90amphi-α allow broader flexibility for client recognition.

“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.”

May combine two claims.

Hop may first prepare Hsp90 for Hsp70 and client interaction, and then rearrange after Hsp70SNBD binding.

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

May combine two claims.

In the proposed pathway, Hsp70C captures flexible GRpre-Helix1, causing the next dynamic helix-strand motif to detach and destabilizing the GR ligand-binding pocket.

“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).”

May combine two claims.

GR is partially unfolded and threaded through the Hsp90 lumen.

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

May combine two claims.

Perturbations to GRHelix1 by Hsp70 or the loading complex lead to loss of GR ligand binding, explaining GR inactivation during the chaperone cycle.

“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.”

May combine two claims.

Both positions also cross-link with Hop, indicating that GRpre-Helix1 cross-links with Hsp70C rather than Hsp70S.

“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.”

May combine two claims.

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, suppressing ligand binding.

“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.”

May combine two claims.

A previous optical-tweezer study showed that GRHelix1 is readily detached, correlating with ligand binding loss.

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

• cites [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). sourceNot checked — no full text of the source

May combine two claims.

Hsp90 repurposes one side of its client-binding sites to bind HopDP2, which augments the Hsp90 lumenal client-binding site and facilitates client loading from Hsp70.

“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).”

Probably not a substantial claim: may not pass S3 (load-bearing).

The range of fitted atomic B-factors accurately reflects the distribution of local-resolution estimates in the global reconstruction.

“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.”

Classified as probably not a substantial claim.

Cross-linking was done right after the complex was fractionated from SEC to ensure that pBpa-Hop and pBpa-GR cross-link the bound segment within the loading complex.

“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).”