Peer review of: Functional 3D architecture in an intrinsically disordered E3 ligase domain facilitates ubiquitin (2019)


REVIEWER COMMENTS 

 

Reviewer #1 (Remarks to the Author): 

 

The manuscript is considerably improved with the additional data strengthening their conclusions. 
The schematics also assist with interpretation of the figures/manuscript. 

 

I have no further concerns. 



 

Reviewer #3 (Remarks to the Author): 

 

The manuscript is much improved and easier to follow. However, the key criticism remains that 
while the biophysical data is abundant, it is less clear what it reveals about the biology of RNF4. My 
main concern is that the role of the basic region is not as important as the authors are proposing. 
Some of the data that reinforced this concern in the previous version has simply been removed 
(Extended data Fig12, extended data table 4). Meanwhile, the new data such as Extended data figure 
19 isn’t compelling that the basic region is functionally important and critical control data isn’t 
shown (in this case the difference between RNF4 KO and RNF expressing cells). 

 

Regarding the specific points previously raised: 

 

1. On the mechanistic side, there is little explanation or data to explain why the basic residues are 
important for function: What they are doing to facilitate compaction of the SIMs? What are they 
interacting with? There seem to be acidic patches in the sequence. Are they interacting with the 
basic region? 

Here it was asked that the functional significance of the basic residues be addressed. The authors 
provide a new hydropathy plot and one new mutant (∆acidic). This mutant shows different 
behaviour on the analytical gel filtration but by smFRET does not look significantly different to WT, in 
contrast to what the authors state (“modestly increased EFRET”, p8). This does not answer why the 
basic region facilitates compaction. There are simple analyses that could be performed that would 

strengthen the case that the basic and acid regions are functionally importance. For instance, 
looking at the sequence conservation between species should be very revealing. If these regions are 
very important they should be highly conserved both in terms of composition and relative spacing. 

 

2. The ∆basic mutant appears to be important for compaction of the ligase, which brings substrate 
and RING together. However, there is no mechanism presented here that explains why and how. 
There is also no data that physically show that the RING domains are actually close to the substrate 
binding SIMs. Without addressing these questions all we know is that mutating RNF4 perturbs its 
structure and function. 

This comment was intended to ensure that the importance of the basic region in bringing the RING 
and substrate together was tested. The new data presented in Figure 5 strengthens the authors 
arguments and shows that RING and SIMs indeed come together and that the ∆basic mutant 
reduces this behaviour. But what is the expected distance between the 2 dyes for the observed 
EFRET values? This information seems to be very important. Also, it is interesting that the reduction 
in the high-FRET states between WT and ∆basic is ~50 % with and without substrate bound. But why 
is there a general reduction in the high-FRET state when the substrate (tetra-SUMO) is present (32 
instead of 52 % for WT and 17 instead of 25 % for ∆basic)? It seems counterintuitive that substrate-
binding reduces the ability to deliver the substrate to the RING. This should be discussed in the main 
text. 

 

3. On the physiological side, some cellular data would help demonstrate that the decrease in activity 
resulting from mutation of the basic residues is functionally relevant. The authors have previously 
characterized and measured the physiological functions of RNF4 in cells (Tatham et al. (2008); Yin et 
al. (2012)). It is therefore surprising that they didn’t test in this study whether their in vitro findings 
are important for RNF4’s cellular function. 

This was a key point as it asked for some cellular data to demonstrate the physiological importance 
of the biophysical measurements. It is good that there has been an attempt to do this but 
disappointing that the assay has not been performed as in the authors previous works. Specifically, a 
key condition is missing from the data, namely quantification of PML bodies in the RNF4 KO cells. 
Without this it is not possible to say what contribution RNF4 is making to PML degradation in the 
experiment nor how much of this contribution the ∆basic mutant is making. If the effect of the 
∆basic mutant is physiologically relevant, it should be close to the phenotype of an RNF4 knockout. 
In addition, a western blot for PML is missing to, which was performed in their original study (Figure 
7 in: Tatham, M. H., Geoffroy, M. C., Shen, L., Plechanovova, A., Hattersley, N., Jaffray, E. G., Palvimo, 
J. J., and Hay, R. T. (2008) RNF4 is a poly-SUMO-specific E3 ubiquitin ligase required for arsenic-
induced PML degradation. Nature Cell Biology 10, 538-546). 

 

4. SUMO Binding in Extended data Fig 12b looks impaired in the gel shift assay in contrast to what is 
stated in the main text (p4 l86 “retained SUMO chain binding”). – It seems more correct to replace 
the original data and explain in the manuscript why it conflicts with other datasets. 

 

5. The table in extended data table 4 suggests that the end-to-end distance is relatively similar 
between WT and ∆basic. (8.4 vs. 10.1). These values are both within the standard deviation of the 
measurements. Is there now a significant opening in the ∆basic mutant or not? It is good that new 
analysis has been added but statements like “The RNF4N ∆Basic mutant displayed a clear reduction 
in loop and hairpin structures… (Fig. 4F and Supplementary Fig, 16)” on page 9 should be avoided 
without statistically significant quantification. 

 

6. The NMR experiments with N- or C-terminal spin-labels hardly show an effect. Why are only 
residues 74 and 75 slightly affected and what about the rest of the construct? – To make the NMR 
clearer to readers, please provide all spectra in the supplemental part. In addition, it is important to 
state which residues were lost and could thus not be analysed (in Methods or an additional Table). 

 

7. The manuscript is very short and there is no discussion. How do the results change our view of 
RNF4 mechanism and function? How does this contrast with our understanding of other ligases? For 
instance, one interesting comparison might be Cullin-RING E3s and how they achieve substrate 
ubiquitinationH (Substrate adaptor on one end, RING on the other, activation by Nedd8 so that the 
RING can span the distance to the substrate (e.g. Duda et al. (2008) Cell). – The new article format 
improves the quality of the manuscript. Although the comparison to Cullin-RING-ligases is useful, 
they are very different to ssRINGs, as the authors also stated. Yet, while Beak et al showed how 
Neddylation effectively brings RING and substrate together, a clear take-home message from this 
RNF4 work is lacking. Rather than simply state that the data “highlights how our finding with RNF 
could offer insight”, please explain what the insight is. 

 

8. Generally this manuscript makes it hard to find information about constructs and more. The 
Figures are not intuitive and proper labelling would make it much easier to understand them (e.g. 
the little cartoons above the PRE NMR data have the wrong size and are therefore confusing. These 
cartoon in the correct size could show where features are in the histogram.) – The revised version of 
the manuscript has much clearer figures. 

 

Due to the new article format of the revised manuscript, additional issues have appeared in the new 
text: 

 

1) The second sentence in the abstract “Structural disorder is prevalent within ssE3s particularly in 
substrate binding and domain linking regions” and third sentence in the second paragraph on page 2 
“Structural disorder is high in ssE3s, with about 25% of all residues predicted to reside in disordered 
regions. These disordered regions are mainly located in the substrate binding domains and inter-
domain linkers, with the ordered RING domain responsible for E2 binding (9)” implies that most 

ssE3s of the RING type would have a high disorder. This in contrast to the fact that the disorder 
distribution within E3s is no different to the rest of the human proteome, as shown in the paper the 
authors reference (Bhowmick, P., Pancsa, R., Guharoy, M., and Tompa, P. (2013) Functional diversity 
and structural disorder in the human ubiquitination pathway. PloS one 8, e65443). Structural 
disorder is thus not prevalent within ssE3s but rather similar to proteins in general. It is true though 
that there is a subset (17.5 % of ssRINGs or 8.5 % of all E3s) of ssE3s with very high disorder (>50 %) 
to which RNF4 belongs. This means that RNF4 is not a general representative of ssRINGs. This 
sentence needs to be altered. 

 

2) The sentence “Molecular dynamics simulations were applied and intramolecular diffusion deemed 
responsible for closing the distance between bound substrate and E2 (9)” implies that the question 
of substrate ubiquitination by CBL was solved by the compaction modelling in MD simulations. 
However, in these simulations, substrate and E2 active site get no closer than 20 Å apart (Bhowmick, 
P., Pancsa, R., Guharoy, M., and Tompa, P. (2013) Functional diversity and structural disorder in the 
human ubiquitination pathway. PloS one 8, e65443). The molecular breathing in MD simulations is 
therefore not responsible for bringing E2 active site and substrate in range for catalysis. This is also 
in line with Cullin-RING-ligases. If molecular breathing alone was responsible, there would not be 
any need for Nedd8 to bring RING, E2 and substrate together (which, as pointed out by the authors, 
is indeed a requirement: Baek, K., Krist, D. T., Prabu, J. R., Hill, S., Klugel, M., Neumaier, L. M., von 
Gronau, S., Kleiger, G., and Schulman, B. A. (2020) NEDD8 nucleates a multivalent cullin-RING-UBE2D 
ubiquitin ligation assembly. Nature 578, 461-466). This sentence needs to be altered. 

 

3) In the first sentence on page 9, the authors claim the following: “resulting in the observed 
compaction in RNF4N ∆Acidic”. This is a strong exaggeration of the observed data. While the 
analytical gelfiltration hints into this direction, the modest increase of EFRET 0.68±0.17 to 0.72±0.17 
does not justify this claim. 

 

Reviewer #4 (Remarks to the Author): 

 

The authors have satisfactorily addressed all of my concerns. I would recommend the publication of 
the manuscript.