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

Reviewers' comments: 

 

Reviewer #1 (Remarks to the Author): 

 

 

The manuscript by Murphy et al (Ron Hay and coworkers) reports an important discovery regarding 
the architecture of a RING E3 ligase that suggests how substrate recruitment domains can be 
spatially positioned in close proximity to the RING domain. Importantly this paper suggests that 
intrinsically disordered regions can have a key role in domain organisation. 

 

The manuscript is well written, concise and highlights the key findings. It also makes a key 
contribution to the field as it is likely that other E3s might have similar properties – this is something 
that it will be important to test. 

 

As a result publication is supported. 

 

Main point: 

1) Previsouly the authors have shown that dimerisation of the RNF4 RING domain is important for 
activity. Have the authors evaluated the impact of RING dimerisation on the compaction of RNF4? It 
would be nice to see how an obligate RNF4 monomer behaves in these assays? 

 

It seems that a constitutive monomeric form of RNF4 would also be compact prior to addition of 
SUMO chains but this will be important to understand as it will inform the order of events associated 
with activation of RNF4. 

 

2) Furthermore, is the SUMO chain ubiquitylated in an intramolecular manner? It seems that this 
model would predict that to be the case but it would be nice if the authors could demonstrate this. 

 

Minor point: 

It might be helpful if the authors included a schematic to sum up their data. 

 

 

 

Reviewer #2 (Remarks to the Author): 

 

The authors employ single-molecule FRET and NMR to study the conformation and flexibility of a 
protein linker of the E3 ligase RNF4N in the context of ubiquitin transfer. For a substrate recognition 
domain previously thought to be intrinsically disordered, the authors report structurally defined 
conformations. This defined structural architecture might indicate a general mechanism of this 
family of E3 ligases. 

First, I have a couple of questions regarding fluorophore labeling of the RNF4N peptides. According 
to the methods section, peptides were purified using filtration columns, which will remove the 
excess dye. However, this purification will also retain the unlabeled peptide. Did the authors 
determine the degree of labeling for the peptides? And, do I correctly interpret that “stochastic 
labeling” (Methods) refers to the random labeling of dual-cysteine mutants of the RNF4N peptides 
with either fluorophore? I would expect a significant fraction of peptides labeled with two donors 
and two acceptors (in theory, each would be 25%); did the authors analyze the distribution of dual-
labeled peptides? 

Second, I ask the authors to include more details on the FRET analysis. How were single-molecule 
sports pre-selected? The authors averaged intensities from the first ten frames; an alternative (or 
additional) analysis would plot the FRET values for each 100 ms interval, and for complete 
trajectories until acceptor/donor photobleaching. 

Third, the authors show smFRET trajectories of the WT peptide (Fig 1E and SI figures). There seems 
to be no apparent dynamics (in a sense of conformational transitions resulting in distance changes); 
however, the width of the FRET populations (Fig 1C) seems to be different, which could report on 
some intrinsic flexibility in part of the regions. This information could be extracted in addition, and 
would allow to refine the structural model. 

Minor issues 

Please use the term “Förster resonance energy transfer”, according to IUPAC recommendation, 
https://doi.org/10.1351/pac200779030293 

Figure 1C, do the numbers in brackets indicate the position of the fluorophore labels? 

Single-molecule FRET experiments operate at low concentrations compared to biomolecular 
interaction strengths. What concentrations were used in the single-molecule assay? Can the authors 
comment on the K_D of SUMO binding the peptide? One could also think of ensemble FRET 
experiments at concentrations as high as in the shift assays. 

 

 

 

 

Reviewer #3 (Remarks to the Author): 

 

This manuscript from Murphy et al. aims to understand how SUMO-chains are being ubiquitinated 
by the E3 ligase RNF4. This follows their long-standing biological (Tatham et al. (2008); Yin et al. 
(2012)) and mechanistic (Plechanovova et al. (2011), Plechanovova et al. (2012); Xu et al. (2014)) 
interest in the actions of the SUMO-targeted RNF4. 

 

In this particular work, the authors use FRET to show that SUMO binding does not induce substantial 
conformational change in the SIM domains of RNF4 upon binding (Figure 1). The authors also show 
by FRET that mutation of basic residues between the SIM domains and the RING causes the protein 
to be less compact (Figure 2). NMR and MD simulations further support this FRET data (Figure 3). 
Finally, mutation of the basic residues reduces in vitro ubiquitination of a SUMO substrate (Figure 4). 
The biophysical data is of good quality and the fact that the SIMs are ready to engage substrate is of 
interest. 

 

However, although the authors attempt to tackle one of the big questions in the ubiquitin field, 
there are many issues with the presented work. As a study, it feels like one-half of a story and lacks 
detailed mechanistic insight and physiological relevance. 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? 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. There 
seem to be acidic patches in the sequence. Are they interacting with the basic region? Without 
addressing these questions all we know is that mutating RNF4 perturbs its structure and function. 

 

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. 

 

In addition, the following issues were found: 

- 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”). 

- 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? 

- The NMR experiments with N- or C-terminal spinlabels hardly show an effect. Why are only 
residues 74 and 75 slightly affected and what about the rest of the construct? 

- 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). 

- No data in the paper shows that the RINGs are brought in close proximity to the SIMs and thus the 
substrate. 

- 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.) 

 

 

 

Reviewer #4 (Remarks to the Author): 

 

In this manuscript, Murphy et al. showed that a defined architecture within a disordered region 
contributes to E3 ligase function. The authors discovered that the expected disordered substrate 
recognition domain of RNF4 might have a defined conformation before SUMO binding using the 
smFRET and NMR methods. They further mutated the highly basic linker. Using smFRET and 
molecular dynamics simulations, the authors proposed that electrostatic interactions may contribute 
to the formation of the highly flexible but compact structure. This manuscript addressed a significant 
question – what is the mechanism by which SUMO substrate is delivered to the RING - by proposing 
an electrostatic driven compact structure within a disordered region. Overall, I am impressed by the 
quality of the paper. However, the proposed mechanism still suffers from multiple deficiencies. My 
major concerns are that the proposed electrostatic interactions and the global three-dimensional 
architecture are not well defined in the manuscript. Thus, I think the paper is a bit immature to be 
accepted as it is. If the authors could address the points discussed below, it could be a very 
significant and interesting paper. 

 

Major concerns 

 

1. One of the major discoveries of the paper is that “electrostatic interactions involving the highly 
basic region linking the substrate recognition and RING domains juxtaposed those regions and 
mediated substrate ubiquitination”. However, it is not clear what residues the basic region interacts 
with? Is there an acidic region? Or are there any specific acidic residues involved in the electrostatic 
interactions with the basic region? Are these residues located in any specific SIM region? These 
questions are important to define the proposed electrostatic interactions. 

 

2. The smFRET focuses on the conformation of the SIM region and demonstrated that the 
conformation of the SIM region may be primed for SUMO binding. The NMR data, on the other 
hand, suggested that both termini fold back towards the middle of the peptide. From Figure 3D and 
Extended date Figure 19, it seems that not only the basic linker, other regions, including residue 
~105-115 may also have interactions with other residues. But these interactions are not discussed in 
the manuscript. Could the interactions involving residues ~105-115 be important for the highly 
flexible but compact structure? Could the authors use smFRET to measure the conformational 
change between the basic linker and the RING domain (i.e. the C-terminal region, residues 87-133) 
and to show whether the conformation of this region is defined or disordered before and after 
SUMO binding? Or could the mutagenesis experiments demonstrate the roles of residues ~105-115? 

 

3. The authors stated that their studies “have revealed a global three-dimensional architecture 
within this “disordered” region, with highly flexible but compact structures facilitating productive 
binding of SUMO substrate.” However, it is not clear how this 3D architecture is defined. Does the 
entire peptide (residue 32-133) or only SIM+basic linker region (residue 32-88) have a 3D 
architecture? What characterizes the compact structures? 

 

 

4. The molecular dynamics simulations showed the distances between residues 80 and 133 change 
upon the mutation. However, in Extended data Table 4, the authors did not report distances change 
between residues 32-79 upon mutation. Are these distances change significantly upon mutation? If 
not, what could be the reason if the basic residues are critical to maintaining the compact structure 
in the N-terminal region? 

 

Minor Concerns 

 

1. The basic residues are mutated to either serine or alanine. Why are they mutated to different 
residues? 

 

2. On Page 18, “Production trajectories were performed for.” This sentence is not completed. How 
long did the production trajectories were performed for?