Dynamic Aha1 Co-Chaperone Binding to Human Hsp90

Background

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 Hsp90^3,5. 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.

 

Results

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-TROSY^4,10,11,12.

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-chaperones^3,5, 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 (a–c) 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). (d–f) 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.

 

References

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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 https://doi.org/10.1101/550228
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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.
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Tags: aha1, atpase, heat shock proteins, hsp90, methyl-trosy, molecular chaperone, nmr spectroscopy

doi: https://doi.org/10.1242/prelights.8801

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