Structure of Hsp90–p23–GR reveals the Hsp90 client-remodelling mechanism

May combine two claims.

Expression of p23 significantly enhanced GR activity relative to Sba1, consistent with a previous report.

“expression of p23 significantly enhanced GR activity relative to Sba1, consistent with a previous report32.”

• cites [32] Freeman, B. C., Felts, S. J., Toft, D. O. & Yamamoto, K. R. The p23 molecular chaperones act at a late step in intracellular receptor action to differentially affect ligand efficacies. Genes Dev. 14, 422–434 (2000). sourceDisputed

  In “Effects of the yeast p23 homolog on IR activities” (page unavailable), the paper says p23 and Sba1p were “indistinguishable in their effects on receptors.” Its mammalian-cell results report that both increased GR activity two- to threefold, rather than p23 enhancing it significantly more than Sba1p. (LLM, conf=0.88)

  Checked by a reasoning model — may be wrong.

  Experimental — accuracy not yet measured

Classified as probably not a substantial claim.

Hsp90 is an essential conserved chaperone that folds and activates many client proteins.

“Hsp90 is a conserved and essential molecular chaperone responsible for the folding and activation of hundreds of ‘client’ proteins1-3.”

• cites [1] Taipale, M., Jarosz, D. F. & Lindquist, S. HSP90 at the hub of protein homeostasis: emerging mechanistic insights. Nat. Rev. Mol. Cell Biol. 11, 515–528 (2010). sourceNot checked — no full text of the source
• cites [2] Schopf, F. H., Biebl, M. M. & Buchner, J. The HSP90 chaperone machinery. Nat. Rev. Mol. Cell Biol. 18, 345–360 (2017). sourceNot checked — no full text of the source
• cites [3] Taipale, M. et al. Quantitative analysis of HSP90–client interactions reveals principles of substrate recognition. Cell 150, 987–1001 (2012). sourceSupported

May combine two claims.

The kinase:Hsp90 structure showed how Hsp90 stabilizes an inactive client but did not explain how Hsp90 reactivates a client such as GR.

“The kinase:Hsp90 structure11 first revealed how Hsp90 can stabilize an inactive client, but provided no insights to explain how Hsp90 can reactivate a client, such as GR.”

• cites [11] Verba, K. A. et al. Atomic structure of Hsp90–Cdc37–Cdk4 reveals that Hsp90 traps and stabilizes an unfolded kinase. Science 352, 1542–1547 (2016). sourceSupported

May combine two claims.

Hsp90 W320 and F349 also contact GR in the loading complex, and Hsp90 W320 is critical for client activation in vivo.

“Notably, Hsp90W320,F349 also make contact with GR in the loading complex12 and Hsp90W320 is critical for client activation in vivo20-22.”

• cites [12] Wang, R. Y.-R. et al. Structure of Hsp90–Hsp70–Hop–GR reveals the Hsp90 client-loading mechanism. Nature https://doi.org/10.1038/s41586-021-04252-1 (2021). sourceSupported
• cites [20] Meyer, P. et al. Structural and functional analysis of the middle segment of Hsp90: implications for ATP hydrolysis and client protein and cochaperone interactions. Mol. Cell 11, 647–658 (2003). sourcePartly supported

  The Client Protein Binding Sites section (p. 8) reports that a yeast Hsp90 Trp300Ala mutant had severe defects in growth and in vivo v-Src activation, supporting the functional point. Our copy does not identify Trp300 as W320, so it does not directly establish the claim for the specifically numbered residue. The GR-loading-complex contact is attached to a different citation in the citing sentence. (LLM, conf=0.78)

  Experimental — accuracy not yet measured
• cites [21] 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 that yeast Hsp90 W300A reduced GR activation by about 50% in an in vivo reporter assay. The paper studies W300 in yeast and W312 in human Hsp90β, but our copy does not identify or directly test W320; the loading-complex contact claim is attached to a different citation (12). (LLM, conf=0.84)

  Experimental — accuracy not yet measured
• cites [22] 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

  The Results (page unavailable) show that the W300A mutation in yeast Hsp90 strongly affects v-Src activation in vivo, but its effect depends on temperature; GR activity is only moderately affected. Our copy does not identify W300 as W320 or establish that W320 is critical for client activation generally. The separate claim about contact with GR is attached to citation 12, not this source. (LLM, conf=0.83)

  Experimental — accuracy not yet measured

May combine two claims.

Only one p23 is bound to the Hsp90 dimer in the maturation complex, despite excess p23 and two p23 molecules in the yeast structure.

“Only one p23 is bound to the Hsp90 dimer, consistent with a previous report8, although a 2-fold excess of p23 was added during complex preparation and two p23 molecules are in the yeast Hsp90:p23 structure16.”

• cites [8] Lorenz, O. R. et al. Modulation of the Hsp90 chaperone cycle by a stringent client protein. 53, 941–953 (2014). sourceNot checked — no full text of the source
• cites [16] Ali, M. M. U. et al. Crystal structure of an Hsp90–nucleotide–p23/Sba1 closed chaperone complex. Nature 440, 1013–1017 (2006). sourceSupported

May combine two claims.

The Hsp90:p23 interface is comparable to the yeast Hsp90:p23 crystal structure, with p23 contacting Hsp90 NTDs to stabilize the closed state and burying about 1274 Å2.

“The Hsp90:p23 interface is comparable to the crystal structure of the yeast Hsp90:p23 complex (PDB ID 2CG9)16, where p23 makes extensive contacts with the N-terminal domains of Hsp90 (Hsp90NTDs) to stabilize the Hsp90 closed state (~1274Å2 BSA)”

• cites [16] Ali, M. M. U. et al. Crystal structure of an Hsp90–nucleotide–p23/Sba1 closed chaperone complex. Nature 440, 1013–1017 (2006). sourcePartly supported

  The Structural basis section (p. 6) says p23/Sba1 contacts both Hsp90 N-terminal domains and that binding stabilizes the ATP-bound conformation. But our copy does not give the claimed ~1274 Ų buried surface area. The accession-code statement (p. 2) assigns 2CGE to the Hsp90–p23/Sba1 complex and 2CG9 to the separate M-C construct, contrary to the citing sentence’s identification of 2CG9 as the complex. (LLM, conf=0.91)

  Experimental — accuracy not yet measured

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

A peptide corresponding to p23 118-131 was previously found to have helical propensity, and an interaction between p23 117-127 and GR has been suggested.

“Furthermore, a peptide corresponding to this region (p23118-131) was previously found to have helical propensity26 and an interaction between p23117-127 and GR has been suggested27.”

• cites [26] Seraphim, T. V. et al. The C-terminal region of the human p23 chaperone modulates its structure and function. Arch. Biochem. Biophys. 565, 57–67 (2015). sourceNot checked — no full text of the source
• cites [27] Biebl, M. M. et al. Structural elements in the flexible tail of the co-chaperone p23 coordinate client binding and progression of the Hsp90 chaperone cycle. Nat. Commun. 12, 828 (2021). sourceSupported

May combine two claims.

p23 binds and stabilizes the closed Hsp90 conformation and is needed for full reactivation of GR ligand binding in vitro and proper GR function in vivo.

“The cochaperone p23 specifically binds and stabilizes the closed Hsp90 conformation16 and is required for full reactivation of GR ligand binding in vitro10 and proper GR function in vivo17.”

• cites [16] Ali, M. M. U. et al. Crystal structure of an Hsp90–nucleotide–p23/Sba1 closed chaperone complex. Nature 440, 1013–1017 (2006). sourceSupported
• cites [10] Kirschke, E., Goswami, D., Southworth, D., Griffin, P. & Agard, D. 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
• cites [17] 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.e945 (2017). sourceNot checked — no full text of the source

May combine two claims.

Together with the GR-loading complex structure, the authors present the molecular mechanism of chaperone-mediated GR remodeling and the first complete chaperone cycle for any client.

“Together with the GR-loading complex structure12, we present the molecular mechanism of chaperone-mediated GR remodeling, establishing the first complete chaperone cycle for any client.”

• cites [12] Wang, R. Y.-R. et al. Structure of Hsp90–Hsp70–Hop–GR reveals the Hsp90 client-loading mechanism. Nature https://doi.org/10.1038/s41586-021-04252-1 (2021). sourceSupported

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

p23 has general Hsp90-independent chaperoning properties.

“although p23 has general Hsp90-independent chaperoning properties24,30,31.”

• cites [24] Weikl, T., Abelmann, K. & Buchner, J. An unstructured C-terminal region of the Hsp90 co-chaperone p23 is important for its chaperone function. J. Mol. Biol. 293, 685–691 (1999). sourceNot checked — no full text of the source
• cites [30] Weaver, A. J., Sullivan, W. P., Felts, S. J., Owen, B. A. L. & Toft, D. O. Crystal structure and activity of human p23, a heat shock protein 90 co-chaperone. J. Biol. Chem. 275, 23045–23052 (2000). sourceSupported
• cites [31] Freeman, B. C., Toft, D. O. & Morimoto, R. I. Molecular chaperone machines: chaperone activities of the cyclophilin Cyp-40 and the steroid aporeceptor-associated protein p23. Science 274, 1718–1720 (1996). sourceNot checked — no full text of the source

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

The p23 tail-helix was predicted by multiple secondary structure prediction algorithms and AlphaFold v2.0.

“This newly identified p23tail-helix was also predicted by multiple state-of-the-art secondary structure prediction algorithms and AlphaFold v2.0 25”

• cites [25] Jumper, J. et al. Highly accurate protein structure prediction with AlphaFold. Nature 596, 583–589 (2021). sourceSupported

May combine two claims.

GR ligand binding is regulated by a cycle of GR-chaperone complexes in which Hsp70 first inhibits GR, then GR is loaded onto Hsp90:Hop to form an inactive loading complex.

“We demonstrated that GR ligand binding is regulated by a cycle of GR:chaperone complexes in which GR is first inhibited by Hsp70, then loaded onto Hsp90:Hop (Hsp70/Hsp90 organizing protein) forming an inactive GR:Hsp90:Hsp70:Hop loading complex12.”

• cites [12] Wang, R. Y.-R. et al. Structure of Hsp90–Hsp70–Hop–GR reveals the Hsp90 client-loading mechanism. Nature https://doi.org/10.1038/s41586-021-04252-1 (2021). sourceSupported

May combine two claims.

In the loading complex, GR pre-Helix 1 is captured by Hsp70, GR Helix 1 is stabilized by Hop, and GR post-Helix 1 is threaded through the semi-closed Hsp90 lumen.

“In the loading complex, GRpre-Helix 1 is captured by Hsp70, GRHelix 1 is stabilized by Hop, and GRpost-Helix 1 is threaded through the semi-closed Hsp90 lumen.”

• cites [12] Wang, R. Y.-R. et al. Structure of Hsp90–Hsp70–Hop–GR reveals the Hsp90 client-loading mechanism. Nature https://doi.org/10.1038/s41586-021-04252-1 (2021). sourceSupported

May combine two claims.

The p23 tail-helix and GR hydrophobic groove are well conserved, and a helix in the yeast p23 tail supports GR transactivation in vivo.

“In support of this essential p23:GR interaction, the p23tail-helix and GR hydrophobic groove are well conserved, and a helix in the yeast p23 tail supports GR transactivation in vivo27.”

• cites [27] Biebl, M. M. et al. Structural elements in the flexible tail of the co-chaperone p23 coordinate client binding and progression of the Hsp90 chaperone cycle. Nat. Commun. 12, 828 (2021). sourcePartly supported

  The Results sections on p23-tail structure and GR maturation in vivo report a conserved p23 tail helix and show that deleting the yeast helix significantly reduces GR activation in a reporter assay (pages not provided). The cited paper does not establish that a GR hydrophobic groove is well conserved; its discussion of a binding cleft concerns Hsp90, not GR. (LLM, conf=0.86)

  Experimental — accuracy not yet measured

May combine two claims.

Once in the maturation complex, ligand-bound GR is protected from Hsp70 rebinding and re-inhibition, allowing protected nuclear translocation.

“The ligand-bound GR becomes protected from Hsp70 rebinding and re-inhibition once it is in the maturation complex, allowing protected nuclear translocation34,37,38.”

• cites [34] Freeman, B. C. & Yamamoto, K. R. Disassembly of transcriptional regulatory complexes by molecular chaperones. Science 296, 2232–2235 (2002). sourceNot checked — no full text of the source
• cites [37] Czar, M. J., Galigniana, M. D., Silverstein, A. M. & Pratt, W. B. Geldanamycin, a heat shock protein 90-binding benzoquinone ansamycin, inhibits steroid-dependent translocation of the glucocorticoid receptor from the cytoplasm to the nucleus. Biochemistry 36, 7776–7785 (1997). sourceNot checked — no full text of the source
• cites [38] Galigniana, M. D., Radanyi, C., Renoir, J. M., Housley, P. R. & Pratt, W. B. Evidence that the peptidylprolyl isomerase domain of the hsp90-binding immunophilin FKBP52 is involved in both dynein interaction and glucocorticoid receptor movement to the nucleus. J. Biol. Chem. 276, 14884–14889 (2001). sourcePartly supported

  The Results passage on FKBP52 domain I reports steroid-dependent GFP-GR nuclear translocation, and the Discussion links rapid movement to GR–hsp90 heterocomplexes. Neither passage establishes that ligand-bound GR is protected from Hsp70 rebinding or re-inhibition in a maturation complex; searches for those terms found no such mechanism in our copy. Page numbers were not available. (LLM, conf=0.84)

  Experimental — accuracy not yet measured

May combine two claims.

Where examined, SHR activity depends on p23, and the progesterone receptor requires the p23 tail for chaperone-mediated ligand-binding activity.

“Indeed, where examined, SHR activity has been dependent on p2317 and the progesterone receptor (PR) requires the p23 tail for chaperone-mediated ligand-binding activity30.”

• cites [17] 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.e945 (2017). sourceNot checked — no full text of the source
• cites [30] Weaver, A. J., Sullivan, W. P., Felts, S. J., Owen, B. A. L. & Toft, D. O. Crystal structure and activity of human p23, a heat shock protein 90 co-chaperone. J. Biol. Chem. 275, 23045–23052 (2000). sourceSupported

May combine two claims.

Both clients are threaded through the closed Hsp90 lumen, suggesting a universal binding mode for Hsp90 clients.

“Both clients are threaded through the closed Hsp90 lumen, suggesting a universal binding mode for Hsp90 clients11,35”

• cites [11] Verba, K. A. et al. Atomic structure of Hsp90–Cdc37–Cdk4 reveals that Hsp90 traps and stabilizes an unfolded kinase. Science 352, 1542–1547 (2016). sourcePartly supported

  The section “The N-lobe of Cdk4 is significantly unfolded and threads through Hsp90” reports Cdk4 density passing through the Hsp90 lumen; “Hsp90 is in a closed conformation in the complex” establishes that Hsp90 is closed. This paper examines one client, Cdk4, and does not establish that both clients thread through the lumen or that this is a universal binding mode. (LLM, conf=0.88)

  Experimental — accuracy not yet measured
• cites [35] Liu, Y., Elnatan, D., Sun, M., Myasnikov, A. G. & Agard, D. A. Cryo-EM reveals the dynamic interplay between mitochondrial Hsp90 and SdhB folding intermediates. Preprint at https://doi.org/10.1101/2020.10.06.327627 (2020). sourceSupported

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

A split-map approach was used to prevent and monitor overfitting: models were refined against one half map, and the other half was used only for validation.

“Following the split map approach19 to prevent and monitor overfitting, the Rosetta iterative backbone rebuilding procedure was used to refine models against one of the half maps obtained from RELION, with the other half map only used for validations.”

• cites [19] 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.

Sodium molybdate stabilizes Hsp90's closed conformation, probably by mimicking a gamma-phosphate to stabilize the post-ATP hydrolysis transition state.

“20 mM sodium molybdate (used to stabilize the closed conformation of Hsp9011,42,43, likely by acting as a γ-phosphate analog to stabilize the post-ATP hydrolysis transition state of Hsp90 (Extended Data Fig. 2c))”

• cites [11] Verba, K. A. et al. Atomic structure of Hsp90–Cdc37–Cdk4 reveals that Hsp90 traps and stabilizes an unfolded kinase. Science 352, 1542–1547 (2016). sourceSupported
• cites [42] Johnson, J. L. & Toft, D. O. Binding of p23 and hsp90 during assembly with the progesterone receptor. Mol. Endocrinol. 9, 670–678 (1995). sourcePartly supported

  The sections “Molybdate and AMP-PNP Stabilization of p23 Complexes” and “Disruption of p23 Complexes by GA” report that sodium molybdate stabilizes Hsp90-containing complexes and discuss an ATP-like conformational change in Hsp90. They do not identify that conformation as closed or propose γ-phosphate mimicry of a post-ATP-hydrolysis transition state; the paper says the stabilizing mechanism is unknown. Page numbers were not available in our copy. (LLM, conf=0.87)

  Experimental — accuracy not yet measured
• cites [43] Csermely, P. et al. Atp induces a conformational change of the 90-Kda heat-shock protein (Hsp90). J. Biol. Chem. 268, 1901–1907 (1993). sourceNot checked — no full text of the source

May combine two claims.

Interface 2 involves GR Helix 1 packing against the Hsp90B CTD amphipathic helical hairpin and GR Helices 8 and 9 contacting Hsp90B MD/CTD, burying about 488 Å2.

“Interface 2 is mainly comprised of the short GRHelix 1 (GR532-539) packing up against the amphipathic helical hairpin (Hsp90amphi-α) in the C-terminal domain of Hsp90B (Hsp90BCTD) and GRHelices 8 and 9 interacting with Hsp90BMD/CTD (~488Å2 BSA)”

May combine two claims.

Interface 3 involves Hsp90B amphi-α packing against GR Helix 3 and Hsp90A MD residues W320 and F349 contacting GR Helices 4 and 9, burying about 467 Å2.

“In interface 3, the Hsp90Bamphi-α packs against GRHelix 3 and the conserved, solvent exposed hydrophobic residues Hsp90W320,F349, located in the middle domain of Hsp90A (Hsp90AMD), make contact with GRHelices 4 and 9 (~467Å2 BSA)”

May combine two claims.

At 37°C in the absence of SBA1, exogenous expression of full-length human p23 and p23Δtail rescued the sba1 growth defect and showed enhanced viability compared to Sba1.

“At 37°C, in the absence of SBA1 (sba1), exogenous expression of full-length human p23 and p23Δtail rescued the sba1 growth defect and, in fact, exhibited enhanced viability compared to exogenous expression of Sba1.”

May combine two claims.

The structure contains a fully closed, nucleotide-bound Hsp90 dimer with one GR and one p23 on the same side of Hsp90.

“The structure reveals a fully closed, nucleotide-bound Hsp90 dimer (Hsp90A and B) (Extended Data Fig. 2c) complexed with a single GR and a single p23, which occupy the same side of Hsp90 (Fig. 1a,b).”

May combine two claims.

In the GR-maturation complex, binding of a second GR would clash with Hsp90B CTD, although the putative dimerization interface is solvent accessible.

“however, in the GR-maturation complex, while the putative dimerization interface is solvent accessible, the binding of a second GR would clash with the Hsp90BCTD”

May combine two claims.

The reported structure is the highest-resolution Hsp90 structure to date and the first atomic-resolution structure of a client bound to Hsp90 in a native conformation.

“This is the highest resolution Hsp90 structure to date and the first atomic resolution structure of a client bound to Hsp90 in a native conformation”

May combine two claims.

In the maturation complex, GR adopts an active folded conformation despite being bound to Hsp90, with Cα RMSD 1.24 Å to crystal structure PDB 1M2Z.

“Surprisingly, in the maturation complex, despite being bound to Hsp90, GR adopts an active, folded conformation (Cα RMSD of 1.24Å to crystal structure Protein Data Bank (PDB) ID 1M2Z23).”

• cites [23] Bledsoe, R. K. et al. Crystal structure of the glucocorticoid receptor ligand binding domain reveals a novel mode of receptor dimerization and coactivator recognition. 110, 93–105 (2002). sourceNot checked — no full text of the source

May combine two claims.

The NCoA3 motif FNSMMNQM aligns with the p23 tail-helix sequence FSEMMNNM and contains key conserved hydrophobic residues that interact with GR.

“The identified NCoA3 motif (FNSMMNQM) aligns with the p23tail-helix sequence (FSEMMNNM) and contains the key conserved hydrophobic residues that interact with GR”

May combine two claims.

p23 makes direct extensive contacts with GR through the early part of its C-terminal tail (residues 112-133), burying about 702 Å2, while residues 134-160 are not visible.

“Unexpectedly, p23 also makes direct and extensive contacts with GR (~702Å2 BSA) through the early part (p23112-133) of its ~58 residue C-terminal tail (p23103-160), while the following 27 tail residues (p23134-160) were not visible.”

May combine two claims.

Unlike the crystal structure, GR Helix 12 in the maturation complex is not stabilized by a co-activator peptide, although co-activator peptide binding would be sterically allowed.

“Unlike the crystal structure, GRHelix 12 is not stabilized by a co-activator peptide, although co-activator peptide binding would be sterically allowed”

May combine two claims.

GR and Hsp90 form three major interfaces: Hsp90 lumen with GR pre-Helix 1; Hsp90 MD/CTD with GR Helix 1; and Hsp90 MD/CTD with GR Helices 3, 4, and 9.

“GR and Hsp90 have three major interfaces (Fig. 1c-e): (1) Hsp90 lumen:GRpre-Helix 1; (2) Hsp90MD/CTD:GRHelix 1, and (3) Hsp90MD/CTD:GRHelices 3, 4, and 9.”

May combine two claims.

A 13-residue helix in the p23 tail (residues 119-131) binds GR, contrary to prior belief that the tail is unstructured.

“Although the tail was previously thought to be unstructured24, we found a 13-residue helix (p23119-131, p23tail-helix) bound to GR”

• cites [24] Weikl, T., Abelmann, K. & Buchner, J. An unstructured C-terminal region of the Hsp90 co-chaperone p23 is important for its chaperone function. J. Mol. Biol. 293, 685–691 (1999). sourceNot checked — no full text of the source

Classified as probably not a substantial claim.

For Fig. 2d and Extended Data Fig. 7b, the p-value for p23 versus no p23 was less than 0.0001.

“p(p23 vs. no p23) = <0.0001”

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

For Fig. 2d and Extended Data Fig. 7b, the p-value for p23 versus p23Δtail was 0.1512.

“p(p23 vs. p23Δtail) = 0.1512”

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

GR ligand-binding behavior was affected by buffer conditions.

“GR ligand binding behavior was affected by buffer conditions; therefore, reactions were always normalized such that each reaction had equivalent amounts of buffer reagents.”

Classified as probably not a substantial claim. The quote only partly states this claim.

The best focused GR:p23tail-helix classes were combined and refined to a nominal resolution of 2.56 Å.

“The best focused classes were combined (140,217 particles) and refined to a nominal resolution of 2.56 Å.”

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

For Extended Data Fig. 7e, the p-value for p23Δtail versus p23Δhelix-tail was 0.9721.

“p(p23Δtail vs. p23Δhelix-tail) = 0.9721”

Classified as probably not a substantial claim.

For Extended Data Fig. 7e, the p-value for p23 versus p23Δhelix-tail was 0.0021.

“p(p23 vs. p23Δhelix-tail) = 0.0021”

May combine two claims.

The authors present the first atomic-resolution structure of a client bound to Hsp90 in a native folded conformation and the highest-resolution structure of full-length Hsp90 to date.

“We present the first atomic resolution structure of a client bound to Hsp90 in a native folded conformation, as well as the highest resolution structure of full-length Hsp90 to date.”

May combine two claims.

From the same GR:Hsp90:p23 dataset, the authors obtained reconstructions of Hsp90:p23 at 2.66 Å and MBP:Hsp90:p23 at 3.63 Å, both containing Hsp90 lumen density.

“From the same GR:Hsp90:p23 dataset, we also obtained reconstructions of Hsp90:p23 (2.66Å resolution) (Extended Data Fig. 8a,b) and MBP:Hsp90:p23 (3.63Å resolution) (Extended Data Fig. 9a,b), which both contain Hsp90 lumen density”

May combine two claims.

MBP is partially unfolded in the MBP:Hsp90:p23 complex, with density for its two C-terminal helices missing.

“Notably, MBP is partially unfolded, as density for the two C-terminal helices is missing.”

May combine two claims.

The structure reveals that p23 makes extensive contacts with GR through a previously unappreciated helix in the p23 tail, in addition to stabilizing closed Hsp90.

“While previously thought to be a general cochaperone whose primary function is to stabilize closed Hsp90, our structure reveals that p23 also makes extensive contacts with GR through a previously unappreciated helix in the p23 tail.”

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

For Fig. 2d and Extended Data Fig. 7b, group variances were not significantly different (Brown-Forsythe p = 0.9966).

“Fig. 2d and Extended Data Fig. 7b [Brown-Forsythe test p = 0.9966]”

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

The Hsp90:p23 class was refined to a nominal resolution of 2.66 Å.

“This class was refined after per-particle CTF refinement and beam-tilt correction in RELION to a nominal resolution of 2.66 Å.”

Classified as probably not a substantial claim.

For Fig. 2d and Extended Data Fig. 7b, the p-value for p23 versus p23Δhelix-tail was 0.0002.

“p(p23 vs. p23Δhelix-tail) = 0.0002”

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

For Extended Data Fig. 7e, the p-value for p23 versus Sba1 was 0.0002.

“p(p23 vs. Sba1) = 0.0002”

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

A search for the FXXMMN motif in the human UniProtKB database returned 10 hits, including p23 and NCoA3/SRC-3.

“There were 10 total hits on the motif, which included p23 and NCoA3/SRC-3.”

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

The best focused MBP classes were combined and refined to a nominal resolution of 3.63 Å.

“The best focused 3D classes were combined (31,556 particles) and refined to a nominal resolution of 3.63 Å after per-particle CTF refinement and beam-tilt correction in RELION.”

May combine two claims.

In the maturation complex, GR threads through the closed Hsp90 lumen and adopts a native ligand-bound conformation stabilized extensively by Hsp90 and the p23 tail-helix.

“In the maturation complex, GR simultaneously threads through the closed Hsp90 lumen and adopts a native, ligand-bound conformation that is extensively stabilized by both Hsp90 and the p23tail-helix.”

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

The FSC improved slightly at medium resolution (5-10 Å) over the pre-refined reconstruction, but the nominal resolution at 0.143 FSC did not change.

“Although the FSC showed slightly improvement over the pre-refined reconstruction at medium resolution range (5–10 Å), the nominal resolution at 0.143 FSC remained unchanged.”

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

For Extended Data Fig. 7e, group variances were not significantly different (Brown-Forsythe p = 0.1696).

“Extended Data Fig. 7e [Brown-Forsythe test p = 0.1696]”

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

For Extended Data Fig. 7c, group variances were not significantly different (Brown-Forsythe p = 0.9224).

“Extended Data Fig. 7c [Brown-Forsythe test p = 0.9224]”

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

For Extended Data Fig. 7e, the p-value for empty vector versus p23 was 0.0001.

“p(e.v. vs. p23) = 0.0001”

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

For Extended Data Fig. 7e, the p-value for p23 versus p23Δtail was 0.0164.

“p(p23 vs p23Δtail) = 0.0164”

May combine two claims.

Prior work from the authors showed that Hsp70 inhibits GR ligand binding and that Hsp90 with p23 restores it.

“Previously, we revealed GR ligand binding is inhibited by Hsp70 and restored by Hsp90, aided by the cochaperone p2310.”

• cites [10] Kirschke, E., Goswami, D., Southworth, D., Griffin, P. & Agard, D. 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 p23 tail-helix contacts the C-terminal strand of GR (H775) and may allosterically stabilize GR Helix 12 in the agonist-bound position.

“The p23tail-helix also contacts the C-terminal strand of GR (GRH775), potentially allosterically stabilizing the dynamic GRHelix 12 (GR750-767) in the agonist-bound position”

May combine two claims.

The enhanced viability depends on the p23 tail-helix, because p23ΔhelixΔtail abolished it, indicating the p23 tail-helix is functionally important in vivo.

“The enhanced viability is dependent on the p23tail-helix, as p23ΔhelixΔtail abolished this enhancement, indicating the p23tail-helix is functionally important in vivo.”

Classified as probably not a substantial claim.

The native ligand-bound GR LBD is known to dimerize for function.

“The native, ligand-bound GR LBD is known to dimerize for function23”

• cites [23] Bledsoe, R. K. et al. Crystal structure of the glucocorticoid receptor ligand binding domain reveals a novel mode of receptor dimerization and coactivator recognition. 110, 93–105 (2002). sourceNot checked — no full text of the source

May combine two claims.

Hsp90 is a constitutive dimer that opens and closes upon ATP binding, and cochaperones regulate this cycle.

“Particularly, Hsp90 functions as a constitutive dimer that undergoes an open-to-closed transition upon ATP binding and this conformational cycle is further regulated by cochaperones15.”

• cites [15] Krukenberg, K. A., Street, T. O., Lavery, L. A. & Agard, D. A. Conformational dynamics of the molecular chaperone Hsp90. Q. Rev. Biophys. 44, 229–255 (2011). sourceNot checked — no full text of the source

May combine two claims.

The GR hydrophobic patch at the p23:GR interface is conserved across SHRs, which are thought to undergo similar Hsp90/Hsp70 regulation.

“The GR hydrophobic patch at the p23:GR interface is also conserved across SHRs, which are thought to undergo similar regulation by Hsp90/Hsp70 (Extended Data Fig. 6c)5.”

• cites [5] Pratt, W. B. & Toft, D. O. Steroid receptor interactions with heat shock protein and immunophilin chaperones. Endocrine Rev. 18, 306–360 (1997).Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

May combine two claims.

p23ΔhelixΔtail did not reduce GR ligand-binding activity as much as omitting p23, indicating the p23 core has a distinct critical role in stabilizing closed Hsp90 in the maturation complex.

“Importantly, p23ΔhelixΔtail did not reduce the GR ligand-binding activity to the same extent as omitting p23, indicating the p23 core plays a distinct and critical role in stabilizing the closed Hsp90 conformation in the maturation complex.”

May combine two claims.

After ATP hydrolysis on Hsp90, Hsp70 and Hop are released and p23 joins to form an active GR:Hsp90:p23 maturation complex that restores GR ligand binding with enhanced affinity.

“Upon ATP hydrolysis on Hsp90, Hsp70 and Hop are released, and p23 is incorporated to form an active GR:Hsp90:p23 maturation complex, restoring GR ligand binding with enhanced affinity.”

May combine two claims.

During the transition, Hsp90 twists to the p23-stabilized closed conformation, likely facilitating client sliding and rearranging the client-binding site to fully enclose GR pre-Helix 1.

“During this transition, Hsp90 twists to the p23-stabilized closed conformation, likely facilitating client sliding and rearranging the client-binding site to fully enclose GRpre-Helix 1”

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

NCoA3 contains a p23 tail-helix-like motif, suggesting other GR coregulators may use this helix motif to bind the hydrophobic groove on GR and compete with p23, potentially facilitating GR release.

“Intriguingly, NCoA3 contains a p23tail-helix -like motif, suggesting other GR coregulators may utilize this novel helix motif to bind the hydrophobic groove on GR and compete with p23, potentially facilitating GR release.”

May combine two claims.

Although overall Hsp90:client interactions are similar, the folding and functional outcomes for the two clients are opposing, demonstrating evolutionarily determined client-specific conformational remodeling by Hsp90.

“Although the overall Hsp90:client interactions are similar, the outcomes for folding and function of these two clients are opposing, demonstrating evolutionarily determined, client-specific conformational remodeling by Hsp90”

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

Every experiment was repeated at least three times.

“All experiments were performed at least three times.”

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

The composite map was used only for presentation in Fig. 1a and was not used for atomic model building or refinement.

“Note that the composite map was only used for presentation in Fig. 1a, but not used in atomic model building or refinement.”

May combine two claims.

The p23 tail-helix is necessary for enhanced GR ligand-binding activity in vitro, so p23 both stabilizes Hsp90 closure and directly contributes to client maturation.

“This p23tail-helix is necessary for the enhanced GR ligand-binding activity in vitro; thus, p23 not only serves as a cochaperone to stabilize the closure of Hsp90, but also directly contributes to client maturation.”

May combine two claims.

MBP is in an apo state, consistent with unfolding of the last two helices that form part of its binding pocket.

“The MBP is also in an apo state, consistent with the unfolding of the last two helices, which form part of its binding pocket”

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

The dissociation constant between GR and F-dex is approximately 150 nM.

“Note that the dissociation constant (KD) between GR and F-dex is ~150 nM10.”

• cites [10] Kirschke, E., Goswami, D., Southworth, D., Griffin, P. & Agard, D. 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.

Ligand-free GR is either too dynamic or is released quickly from the complex.

“suggesting ligand-free GR is either too dynamic or quickly released from the complex.”

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

Because GR Helix 1 slides through the Hsp90 lumen, Hsp90 may act processively to remodel other client domains beyond the one initially engaged, potentially explaining the other reconstructions.

“Given that GRHelix 1 slides through the Hsp90 lumen, it is possible that Hsp90 can act processively to remodel other client domains beyond the one initially engaged, potentially explaining the other reconstructions.”

May combine two claims.

GR pre-Helix 1 can slide into the Hsp90 lumen, allowing GR Helix 1 to refold onto the GR core and generate a ligand-binding-capable native GR stabilized by the p23 tail-helix.

“Then, GRpre-Helix 1 can slide into the Hsp90 lumen, allowing GRHelix 1 to refold onto the GR core, thereby generating a ligand binding capable, native GR, stabilized by the p23tail-helix”

May combine two claims.

The effect of deleting the unstructured part of the p23 tail on GR activity in vivo may be related to p23-dependent regulation of GR functions downstream of ligand binding, which may dominate any effect of the p23 tail-helix on GR activation in vivo.

“The effect of deleting the unstructured part of the p23 tail (p23134-160) on GR activity in vivo may be related to p23-dependent regulation of GR functions downstream of ligand binding34, which may dominate any effect of the p23tail-helix on GR activation in vivo.”

• cites [34] Freeman, B. C. & Yamamoto, K. R. Disassembly of transcriptional regulatory complexes by molecular chaperones. Science 296, 2232–2235 (2002). sourceNot checked — no full text of the source