Dynamic Aha1 Co-Chaperone Binding to Human Hsp90

Javier Oroz, Laura J Blair, Markus Zweckstetter

Posted on: 19 February 2019

Preprint posted on 14 February 2019

Article now published in Protein Science at

Aha! Now I can see you... binding to the Hsp90 chaperone

Selected by Reid Alderson

Categories: biochemistry, biophysics


Seemingly disparate diseases such as type II diabetes, cystic fibrosis, ALS, and Alzheimer’s share a common molecular hallmark, the misfolding of proteins1. However, cells have armed themselves with an evolutionarily conserved defense mechanism to combat protein misfolding: a class of proteins known as molecular chaperones that can help nascent proteins fold, refold incorrectly folded proteins, or recognize misfolded proteins and target them for degradation2. A key molecular chaperone is the 90 kDa heat shock protein (Hsp90), which is present in all eukaryotes and essential for cell viability3. A recent preLight on the molecular chaperone activity of Hsp90 can be found here.

Hsp90 is an ATP-dependent chaperone and therefore requires ATP hydrolysis for function3. In order to efficiently utilize the energy liberated from ATP hydrolysis for its molecular chaperone activity, the ATPase activity of Hsp90 is tightly regulated. The structural plasticity of Hsp90 provides such regulation:  in its unbound form, the Hsp90 dimer populates an open V-shaped, with either ATP binding alone or the combined binding4 of co-chaperone and ATP promoting a transition to a closed conformation3. Furthermore, the Hsp90 dimer dynamically populates an equilibrium between multiple states (open, closed, intermediate), with co-chaperones and substrates shifting the equilibrium5. Over 20 Hsp90-interacting co-chaperones have been identified and these co-chaperones regulate the activity and substrate binding of Hsp903,5. Of these co-chaperones, however, only Aha1 is capable of stimulating Hsp90’s ATPase activity, signifying its crucial role as a regulator of Hsp90 activity.

Here, in this preprint6, the authors utilized nuclear magnetic resonance (NMR) spectroscopy to interrogate the interaction between human Hsp90 (HSP90β) and its co-chaperone Aha1. NMR spectroscopy is exquisitely sensitive to biomolecular interactions and molecular motions (see preLight here), rendering it a highly effective tool for the study of chaperones and their dynamic, plastic nature7. Due to the conformational gymnastics of Hsp90, many previous structural studies employed truncated constructs to simplify data collection and analysis. However, the preprint authors here make use of the full-length protein, thereby providing a more wholistic view.



Previously, a crystal structure had been solved8 of a complex between the N-terminal region of Aha1 and the middle region of Hsp90. Structural data for the interaction between the two full-length proteins remained elusive and thus, since ATP is hydrolyzed in the N-terminal domain of Hsp90, it was unclear how Aha1 stimulated the ATPase activity of Hsp90. Here, the authors6 utilized the full-length proteins and NMR spectroscopy to gain a detailed and complete picture of this important interaction.

Because of the large size of the Hsp90-Aha1 complex (ca. 220 kDa), traditional NMR methods would be intractable due to very rapid NMR signal decay that hinders the study of molecules larger than ca. 50-60 kDa. Instead, the authors made use of a specific type of NMR spectroscopy called methyl-TROSY, which allows the study of biomolecules in excess of 1 MDa9. Methyl-TROSY requires 2H-labeled proteins that have selectively 1H, 13C-labeled methyl groups, which enables preservation of the NMR signals due to the favorable spectroscopic properties of methyl groups. Thus, only the methyl-bearing residues that have been isotopically labeled can yield NMR signals, lowering the number of probes for structural insight but also reducing spectral overlap. The authors here use isoleucine-labeled Hsp90, similar to prior studies of human Hsp90 by methyl-TROSY4,10,11,12.

With methyl-TROSY, the authors6 identified the Aha1 binding site on Hsp90, including widespread structural changes to the N-terminal domain of Hsp90, the site where ATP binds and gets hydrolyzed. When studying a biomolecular interaction by NMR, changes to NMR signals upon binding of a partner can manifest either from the direct binding event or from allosteric structural changes. It is well known that Hsp90 undergoes widespread structural changes upon binding to co-chaperones3,5, with allosteric rearrangements facilitating an “open” to “closed” transition. To disentangle the changes due to binding versus those from allostery, the authors made use of a truncated Hsp90 construct (Hsp90NM) lacking the C-terminal domain, which exists solely as a monomer. This allowed the authors to identify the outer area of the N-terminal domain (distant from the dimerization site) as an Aha1 binding site6.


Figure 3 from the preprint. Shown in (ac) are zoomed-in nuclear magnetic resonance (NMR) spectra (2D 1H-13C HMQC) of methyl-labeled Hsp90 (Ile-δ1 methyl groups are 1H, 13C labeled) in the (a) absence of nucleotide (red) bound to the co-chaperone Aha1 (green), which is NMR-invisible; (b) presence of ADP (purple) bound to the co-chaperone FKBP51 (gold), which promotes an open/extended conformation of Hsp90; or (c) presence of a non-hydrolyzable ATP analogue (AMP-PNP) (red) bound to FKBP51 (blue). (df) Small-angle X-ray scattering (SAXS) pair-distance distribution functions, P(r), for (d) Hsp90 (red), Hsp90-Aha1 (green), or Hsp90-Aha1 in the presence of AMP-PNP (cyan); (e) Hsp90 in the presence of ADP (gold) or Hsp90-FKBP51 in the presence of ADP (purple); and (f) Hsp90 in the presence of AMP-PNP (red) and FKBP51 (blue). This figure is reproduced here under a CC-BY-NC-ND 4.0 International license.


But how does Aha1 binding facilitate increased ATPase activity in Hsp90? An elegant study using yeast Hsp9013 reported both symmetric (2:2 Aha1:Hsp90) and asymmetric (1:2 Aha1:Hsp90) interactions involving Aha1 and Hsp90. Likewise, the stoichiometry of the interaction between co-chaperone p23 and Hsp90 was reported to be either a 1:2 ratio14 or 2:2 ratio4 of p23:Hsp90. NMR spectra are highly sensitive to symmetry, and the NMR data in this preprint are consistent with a symmetric interaction between two Aha1 molecules and the Hsp90 dimer6. A lowly populated asymmetric interaction cannot be ruled out, but the major state appears symmetric based on the single set of NMR signals that are present (e.g. Fig. 1b in the preprint). Finally, it is known that Hsp90 must transition to a closed conformation, placing the N-terminal domains in contact, for ATPase activity to be stimulated. The authors’ NMR data suggest that regions in the N-terminal domain undergo structural changes near the dimerization site, but their small-angle X-ray scattering data indicate that Hsp90 remains in a partially open/partially closed state6. Thus, the fully closed state is not formed and so it remains unclear if the N-terminal domains are in contact. Instead the data suggest that Aha1 binding leads to formation of an intermediate state, perhaps with transient contacts between N-terminal domains.

This study paves the way for future work investigating the inter-molecular contacts of the N-terminal domains of Hsp90 while bound to Aha1, as well as the mechanism of ternary complex formation involving Hsp90, Aha1, and other co-chaperones. Structural characterization of the Aha1-bound, dimer-like conformation of the Hsp90 N-terminal domain will prove insightful in understanding the mechanism of Aha1-induced stimulation of ATPase activity.


Why I chose this preprint

This preprint makes use of NMR spectroscopy to provide detailed insight into the mechanism of the co-chaperone Aha1 and its interaction with Hsp90. The first crystal structure of the truncated Hsp90-Aha1 complex appeared8 already in 2004, but difficulties in obtaining high-resolution data on the full-length complex prevented a wholistic view. Building upon and making use of previously solved crystal structures and prior biochemical and genetic assays, the new NMR data enable a powerful synthesis of data to aid in understanding the regulation of Hsp90 by Aha1.



  1. Chiti F, Dobon CM. (2017) Protein misfolding, amyloid formation, and human disease: a summary of progress over the last decade. Annu. Rev. Biochem. 86: 27-68.
  2. Balchin D, Hayer-Hartl M, Hartl FU. (2016) In vivo aspects of protein folding and quality control. Science 353: aac4354.
  3. Schopf FH, Biebl MM, Buchner J. (2017) The HSP90 chaperone machinery. Nat. Rev. Mol. Cell. Biol. 18: 345-360.
  4. Karagoz GE, Duarte AM, Ippel H, Uetrecht C, Sinnige T, van Rosmalen M, Hausmann J, Heck AJ, Boelens R, Rudiger SG. (2011) N-terminal domain of human Hsp90 triggers binding to the cochaperone p23. Proc. Natl. Acad. Sci. U S A 108: 580-585.
  5. Sahasrabudhe P, Rohrberg J, Biebl MM, Rutz DA, Buchner J. (2017) The plasticity of the Hsp90 co-chaperone system. Mol. Cell 67: 947-951.
  6. Oroz J, Blair LJ, Zweckstetter M. (2019) Dynamic Aha1 co-chaperone binding to human Hsp90. bioRxiv
  7. Burmann BM, Hiller S. (2015) Chaperones and chaperone-substrate complexes: dynamic playgrounds for NMR spectroscopists. Prog. Nucl. Magn. Reson. Spectrosc. 86: 41-64.
  8. Meyer P, Prodromou C, Liao C, Hu B, Roe SM, Vaughan CK, Vlasic I, Panaretou B, Piper PW, Pearl LH. (2004) Structural basis for recruitment of the ATPase activator Aha1 to the Hsp90 chaperone machinery. EMBO J. 23: 1402-1410.
  9. Rosenzweig R., Kay L.E. (2014) Bringing dynamic molecular machines into focus by methyl-TROSY NMR. Annu. Rev. Biochem. 83: 291-315.
  10. Karagoz GE, Duarte AM, Akoury E, Ippel H, Biernat J, Moran Luengo T, Radli M, Didenko T, Nordhues BA, Veprintsev DB, Dickey CA, Mandelkow E, Zweckstetter M, Boelens R, Madl T, Rudiger SG. (2014) Hsp90-Tau complex reveals molecular basis for specificity in chaperone action. Cell 156: 963-974.
  11. Oroz J, Chang BJ, Wysocznski P, Lee CT, Perez-Lara A, Chakraborty P, Hofele RV, Baker JD, Blair LJ, Urlaub H, Mandelkow E, Dickey CA, Zweckstetter M. (2018) Structure and pro-toxic mechanism of the human Hsp90/PPIase/Tau complex. Nat. Commun. 9: 4523.
  12. Oroz J, Kim JH, Chang BJ, Zweckstetter M. (2017) Mechanistic basis for the recognition of a misfolded protein by the molecular chaperone Hsp90. Nat. Struct. Mol. Biol. 24: 407-413.
  13. Retzlaff M, Hagn F, Mitschke L, Hessling M, Gugel F, Kessler H, Richter K, Buchner J. (2010) Asymmetric activation of the Hsp90 dimer by its cochaperone Aha1. Mol. Cell 37: 344-354.
  14. Siligardi G, Hu B, Panaretou B, Piper PW, Pearl LH, Prodromou C. (2004) Co-chaperone regulation of conformational switching in the Hsp90 ATPase cycle. J. Biol. Chem. 279: 51989-51998.








Tags: aha1, atpase, heat shock proteins, hsp90, methyl-trosy, molecular chaperone, nmr spectroscopy


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Author's response

Javier Oroz shared



  1. (R.A.) What is the stoichiometry of the Hsp90-Aha1 binding interaction in your NMR and SAXS experiments? One would expect to see NMR peak doubling if there were an asymmetric interaction (1:2 Aha1:Hsp90), but this does not appear to be the case for either your NMR spectra or those previously obtained11 on Hsp90/Aha1 fragments.


(J.O.) The stoichiometry found previously was Hsp90:Aha1 (2:1), meaning one Aha1 per dimer. In that study they focused on the yeast complex. As you point out, NMR data suggest that we have equimolar stoichiometry in our interactions. Is it because in human proteins rather a symmetric complex is formed? It could be the case. We have examples that the further addition of a different co-chaperone will replace one already bound co-chaperone to establish final asymmetric complexes. Indeed, the model proposed by the mentioned group for the Hsp90:Aha1 complex and ours are not identical. Is it because in our case, we focused on nucleotide-free Hsp90? It could be the case. We wanted to understand the possible scenario in which Aha1 binds before nucleotide to Hsp90, therefore Aha induces certain conformational rearrangements in Hsp90 that facilitate posterior nucleotide binding. However, for clarity, in the models proposed we only include one Aha in the complex with Hsp90 dimer (it does not change the message of the study).


  1. (R.A.) Do you know if the N-terminal domains of Hsp90 contact each other when Aha1 is bound? One would speculate that N-terminal domain dimerization is required for stimulation of ATP hydrolysis, but perhaps Aha1 instead induces a conformational rearrangement near the dimerization site, leading to a “dimer-like” conformation?


(J.O.) According to our SAXS data, the population of closed Hsp90 dimers upon Aha1 binding is still low. That’s why we do not think that the new NMR signals that appear upon Aha1 binding come from Hsp90N dimerization, because the signals are quite strong. We compared these signals to those induced by a different co-chaperone -that blocks Hsp90 in a fully extended conformation– AND nucleotide and we see similar new peaks in the spectra; meaning that these new peaks unlikely come from Hsp90N dimerization in a fully closed dimer.


We propose that the new peaks that appear in Aha1 binding are, as you correctly mention, coming from “dimer-like” structures of Hsp90N induced by Aha1, but still present in a partially open conformation of the dimer. This could be an additional mechanism for triggering the closed conformation of Hsp90 dimer, which, especially for the human protein, is not spontaneous.


In summary, we believe that Aha1 induces “dimer-like” (I like the expression…) changes in Hsp90N domains that will facilitate strong ATP binding and closure of the dimer. However, the interaction of Aha1-C with Hsp90N domains is rather fuzzy because Hsp90N will hydrolyze ATP adopting additional conformations in the following steps of the activation cycle, so Aha1C cannot block a specific conformation of Hsp90N, it needs to be very dynamic.


All these observations strengthen the potential of studying such dynamic complexes by NMR.

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