Peer review of: CryoEM structure of the low-complexity domain of hnRNPA2 and its conversion to pathogenic am (2020)

REVIEWER COMMENTS 

Reviewer #1 (Remarks to the Author): 

This is an interesting article on the fibril structure of the low-complexity domain of hnRNPA2 
examined by cryoEM and X-Ray crystallography. I was asked to assess the crystal structure. The 
structure was determined to high/atomic resolution and the validation reports are excellent, 
indicating the structure is well refined. I also examined sections of the manuscript relevant to the 
crystal structure, e.g. the results, methods, Figure 4, Table 3, and Supplementary Figure 5. I have 
some minor comments: 
1. There appears to be small differences between the table in the validation report and Table 3. 
E.g. completeness and I/sigI. Could the authors please double-check? 
2. The methods refer to Table 5. Should this be Table 3? 
3. Figure 4b would benefit from additional labelling (e.g. of the mating strand and native residues) 
to help readers cross-reference to the text. 
4. In the supplementary figure caption, 'Crystals growth' should be 'Crystal growth' 


Reviewer #2 (Remarks to the Author): 

Overall it is an interesting study about structural aspects of reversible functional fibrils and beside 
the structure it discusses biological relevant aspects. 

My main issues: 
Question 3 at the end of the introduction (line 71) about how a single mutation converts functional 
to pathogenic fibrils is not clearly answered. At least not based on the presented structures. That 
makes also part of the title incorrect. 

Section “The disease-causing mutant core segment”: The hexamer GNYNVF steric zipper crystal 
structure and the cryo EM structure are really hard to compare. There are to many factors that can 
influence the structure and it is difficult to draw conclusions about impact of a mutation by 
comparing the hexameric dimer to a corresponding part in a bigger structure. You would need a 
structure of the mutant D/V hnRNPA2-LCD fibril to get a better understanding of the impact of the 
mutation. 
The comparison between GNYNVF and GNYNDF with the mutant to be more stable than the WT is 
an interesting aspect. GNYNVF and GNYNDF dimer structure comparison could also help finding 
features to explain the different stabilities. 
But for now the hexamer structure doesn’t seem be relevant for the discussion and doesn't give a 
clear answer about the function of the mutation. 

Starting at line 240 regarding polymorphism in pathogenic irreversible amyloid fibrils: 
A recent ex vivo TTR-fibril reconstruction also showed a monomorphic sample and you can adjust 
fibrillization conditions to produce monomorphic in vitro fibrils. You can maybe say most studied 
pathogenic irreversible fibril structures are polymorphic. At the moment it sound like all of them 
are polymorphic. If I understand the authors point correctly functional reversible fibrils need to be 
monomorphic to work, but for pathogenic fibrils it doesn't matter. 

The sentence (line 240-242) “In contrast, structures of pathogenic irreversible amyloid identified 
have shown polymorphism, whether the fibrils were produced ex vivo or in vitro.” I think 
“identified” was meant to be “fibrils”? 

Minor stuff: 
Supplementary figure 1d a better image to show fibrils at 65° or above would be nice. It’s difficult 
to identify fibrils in the current picture. 
I think there is a converting problem for the pdf with the ° sign in the description, I see question 
marks in boxes. Also, there are um instead of µm. 



Reviewer #3 (Remarks to the Author): 

The manuscript by Lu et al. reports the cryoEM structures of fibril formed by hnRNPA2 low-
complexity domain (LCD) fused with mCherry. Unlike the pathological amyloid fibrils, it is 
reversible and displays distinct structural characteristics. Importantly, the Nuclear Localization 
Signal (NLS) of hnRNPA2 was found in the fibril core, which provide explanation on why Kapβ2 
effectively prevents hnRNPA2 fibrilization both in vitro and in cells. In addition, they determined 
the crystal structure of disease-mutation-containing segment of hnRNPA2, and sought to explain 
how a point mutation could switch fibril structure from reversible to irreversible form. Overall, the 
structural data presented to support these findings are generally of high quality. The cryoEM fibril 
structure of hnRNPA2 LCD presents a novel arrangement of reversible fibril. These structures are 
exciting and would be of interest not only to the hnRNPA field but also for the amyloid field in 
general. However, I do have major concerns that need to be addressed and recommended 
suggestions to improve the work. 

Major concerns: 
1. It’s known that amyloid fibril structures are highly polymorphic. Under different circumstances 
(e.g. mutation, PTM, co-factors), the same protein may form distinct fibril structures. The authors 
used the mCherry fused hnRNPA2 LCD to prepare the fibril samples for cryoEM studies. It is 
questionable whether mCherry which contains over 200 residues alters the overall architecture of 
the hnRNPA2 LCD fibril. A recent study demonstrates that the florescence protein (GFP) fused to 
Tau significantly alters the fibril structure of Tau due to the potential steric hindrance 
(https://doi.org/10.1101/2020.03.25.998831). This observation immediately boosts a heated 
discussion in amyloid field (https://www.alzforum.org/news/research-news/widely-used-tau-
seeding-assay-challenged). 
Therefore, in order to strengthen the physiological relevance of the fibril structure reported here, 
it’s important to rule out the possible influence of the fused mCherry in the formation of hnRNPA2 
LCD fibril. The author might need to prepare hnRNPA2 LCD without the mCherry and characterize 
whether it retains the similar fibril structure. I understand that it will take some time given the 
coronavirus outbreak. Rather than resolving the atomic fibril structure of hnRNPA2 LCD alone by 
cryoEM, the authors may use AFM, NS-TEM to compare the fibril structures of the mCherry fused 
LCD and the LCD alone. 

2. The crystal structure of G6F provides very limit yet confusing information in explaining the fibril 
structures and the reversibility of WT and mutated hnRNPA2 LCD fibril. It’s not surprising at all 
that the backbones of the G6F in the crystal fibril and in cryoEM fibril overlay well, since both of 
the segments adopt beta-conformation. The author analyzed the dry interface of the G6F homo-
zipper from the crystal structure and proposed that it may explain the irreversibility of the mutated 
fibril. However, to form this homo-zipper interface, the hnRNPA2 LC subunit needs to totally 
rearrange (due to the steric hindrance) in the protofilament structure. Moreover, the two 
protofilaments need to further intertwine and bundle together. Thus, if the authors want to include 
the homo-zipper structure to explain the LC fibril, they need to provide additional structural 
evidence to support this indeed occurs in the irreversible fibril formed by the mutation rather than 
in G6F crystal. For instance, the authors may measure the thickness of the irreversible mutation 
fibrils, at least to demonstrate that it’s formed by two protofilament rather than a single one 
observed in their reversible fibril of hnRNPA2 LC. Otherwise, the authors might need to remove the 
homo-zipper structure to avoid the confusion and overinterpretation. 

Minor comments: 
1. To fully assess the reversibility of the hnRNPA2 LCD hydrogel, the authors might need to test 
whether the heat-dissolved hnRNPA2 LCD protein can form hydrogel again upon cooling down to 

4℃ with further incubation. 

2. It’s very confusing that the authors directly compared the crystallin form of the mutant segment 
GNYNVF and the fibrillar form of segment GNYNDF, and came up with a conclusion that the mutant 
segment forms more stable structure than the WT segment. These data can only tell the mutant 
segment GNYNVF is more prone to crystallize, while the segment GNYNDF tends to form fibril 
under the condition tested in this study. If the authors want to compare the fibril stability, they 
need to prepare the fibrillar rather than the crystal form of the mutant segment GNYNVF.