Suppression of aggregate and amyloid formation by a novel intrinsically disordered region in metazoan Hsp110 chaperones

Unekwu M. Yakubu, Kevin A. Morano

Preprint posted on 13 January 2021

Hsp110: NEF by day, holdase by night

Selected by Utrecht Protein Folding and Assembly

Categories: biochemistry

Written by: Anna Enneking, Simon Hinfelaar, Inge Rothuis and Vincent Spit


Protein misfolding can cause devastating problems in the cell, as exemplified by the formation of amyloid fibrils in neurodegenerative diseases [1]. Chaperones, including the ATP-dependent Hsp70, prevent protein aggregation [2]. Hsp70 needs a nucleotide exchange factor (NEF) such as Hsp110 to promote cycling from ADP to ATP [3]. Interestingly, Hsp110 is part of the Hsp70 superfamily, meaning they share the canonical Hsp70 architecture. They both have a nucleotide binding domain (NBD) that binds ATP, a flexible linker, and a substrate binding domain (SBD) composed of two parts: a β-sandwich-containing substrate binding site (SBD-β) and α-helical sequence (SBD-α).

Hsp110 also has an extended C-terminus, which is conserved in metazoan Hsp110 genes, e.g human Hsp105α and Apg-1, and has an unknown function [4]. Although they share a canonical structure, Hsp110 has no refolding activity as Hsp70 does. However, Hsp110 overexpression can prevent aggregation of the huntingtin protein in a Drosophila model system [5]. It is unknown whether this is a function of Hsp110 as a NEF or as a passive holdase. To understand this, the authors decide to disrupt the Hsp110 holdase function by targeting the SBD-β domain, because they suspect that this domain is responsible for the holdase activity. In their attempts to disrupt the SBD-β, they uncover a secondary binding site.


Key findings

The authors introduce different deletions in the SBD-β domain of Drosophila Hsp110, leaving the regions that interact with Hsp70 (NBD, SBD-α) intact. They monitor protein aggregation by a light scattering aggregation assay using citrate synthase as substrate to test the holdase activity. Surprisingly, the Hsp110 protein in which SBD-β is deleted is more efficient in preventing aggregation than the full-length protein. So, if the SBD-β is not essential for the activity in this assay, then what else is?

The authors now suspect intrinsically disordered regions (IDRs), as such regions can act as sites for protein binding and are present in many chaperones [6]. In silico analysis reveals one IDR in the C-terminus and another one in the SBD-α domain. It turns out that it is the C-terminal IDR that is responsible for the holdase activity, because when deleted the holdase activity is lost. Deletion of the SBD-α domain does not affect the holdase activity. The human homologs of Hsp110, Apg1a and Hsp105α, also have an IDR in the C-terminus. These C-termini also prevent protein aggregation when directly linked to the NBD (Fig. 4C), so the presence of an IDR in the C-terminus could indicate a general holdase function in the Hsp110 proteins.

Figure 4C and 5C of the preprint: The C-terminal IDR is key for Hsp110 holdase activity. 4C: Light scattering assay showing inhibition of aggregation by the Hsp110 variants. 5C: Negative staining TEM monitoring the effect of Hsp110 variants on amyloid formation of Aβ42. Reproduced with permission from the authors.


The authors now study whether the IDRs can also prevent amyloid formation, which is associated with various diseases including Alzheimer ‘s [1]. To this end, they examine the impact of variants of Drosophila Hsp110 (dHsp110) and its human homolog Hsp105α on fibril formation of amyloid-β 42 (Aβ42).  Negative staining TEM shows that NBDdHsp110 alone does not decrease Aβ42 fibril formation, but the IDR fusions NBDdHsp110-IDRdHsp110 and NBDdHsp110-IDRHsp105α do (Fig. 5C). Thus, the IDR in Hsp110 homologs can also prevent amyloid formation.


What we like about the preprint

We were directly drawn to this article when we read the title. We found the fact that Hsp110 can prevent protein aggregation very interesting, mainly because we previously learned that Hsp110 works as NEF of Hsp70. It was therefore intriguing to read that Hsp70 and Hsp110 share a canonical structure, but that the SBD-β in Hsp110 is not solely responsible for its holdase activity. It was surprising to learn that an IDR contributes to this activity. To our excitement, this paper shows that the IDR in Hsp110 can actually prevent amyloid formation, which is associated with neurodegenerative diseases. This knowledge is important to better understand how chaperones prevent this pathogenic process in the cell.

Tags: amyloid fibrils, chaperones, hsp110, protein aggregation

Posted on: 14 April 2021 , updated on: 21 April 2021

doi: Pending

Read preprint (1 votes)

Author's response

Kevin Morano shared

The full length Hsp110 exhibited less potent holdase activity than the hybrid NBD-C-term protein, without the SBD domain. Do you think it is possible that Hsp110 first evolved as a ‘regular’ holdase chaperone with its IDR and that its main function as NEF evolved later?

From what we can gather from studies in yeast it appears the NEF activity of Hsp110 is the predominate function in the cell.  Mutations to the Sse1 SBD-β disrupt substrate binding activity in vitro; however, these mutations had a mild effect on yeast growth.  On the other hand, mutations disrupting NEF activity exhibited deleterious effects on yeast growth and ability to deal with proteotoxic stress.  It appears substrate binding is expendable for Sse1 function in vivo.  Based on these previous findings from our group we think NEF activity evolved earlier and is the more important of the two Hsp110 activities.

In the TEM experiments NBDdHsp110-IDRHsp105α formed small globular structures, but the homologous NBDdHsp110-IDRdHsp110 did not. Do you have an idea why?

We are not sure why NBD-IDRHsp105α forms these globular structures.  We assume the IDRs of this fusion protein are interacting and driving the macromolecular organization, as the isolated NBD does not have this property. As we state in the paper, their size and ultrastructure are strikingly similar to that of αB-crystallin chaperones, which also contain IDRs.  We also note that  the IDR regions of metazoan Hsp110s are not strictly conserved; sequence divergence may explain why the human, but not fly, NBD-IDR constructs formed these globular structures in vitro. More importantly, we have no reason to ascribe a specific biochemical function to the structures as both the fly and human NBD-IDR fusions chaperoned similarly. Something to pursue!

The manuscript was recently published in the Journal of Biological Chemistry. What were the main changes you made as a result of the peer-review process?

Luckily, there were only a few changes needed before JBC accepted the paper for publication.  To assist readers in understanding the various fusion and deletion constructs utilized, we were asked to add some cartoon schematics  and we answered specific reviewer questions about some of the constructs we did not include in the final version of the paper.  We showed that removing the C-terminal extension alone or in combination with SBD-α did not disrupt Hsp110 substrate binding activity, confirming previous findings that SBD-β is a bona fide holdase domain.  One reviewer was concerned that our slightly lower total salt concentration might have contributed to our new findings. We therefore repeated a few of our experiments in a higher salt buffer and found no reproducible difference in chaperone behavior, negating that issue. We were very gratified to see significant attention paid to the story as a pre-print on BioRxiv, including multiple retweets from neurodegenerative disease investigators and research foundations.

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