Peer review of: Flexibility of intrinsically disordered degrons in AUX/IAA proteins reinforces auxin co- (2019)

Reviewers' comments: 

Reviewer #1 (Remarks to the Author): 

This manuscript combined an impressive array of methods to characterize the interaction between 
two AUX/IAA proteins with TIR1 in the context of auxin sensing. The authors identified new 
binding interfaces between AUX/IAA and TIR1 that were not present in the crystal structures in the 
2007 Nature paper. Crosslinking MS experiments and mutagenesis data nicely corroborate to 
support the importance of the new binding sites, which allowed the authors to propose a structural 
model of the complex. The biochemical data are very solid and the findings are highly novel. These 
findings provided structural basis for understanding the differential auxin binding of the co-
receptor complexes with different AUX/IAA components. However, a major caveat of the 
manuscript is that the paper solely relied on yeast two hybrid binding assays and in vitro data. 
Therefore, the manuscript would be much strengthened if the authors can show that the identified 
new interfaces actually affect AUX/IAA degradation in response to auxin in cells. For example, the 
mutations of TIR1 (D170K and S172A) t dramatically abolished binding of AUX/IAA in Y2H 
experiment, do they affect AUX/IAA degradation at all ? Same questions could be asked for the 
AUX/IAA chimera or deletion mutants. Adding the functional data would make the paper more 
relevant to plant genetic studies. 

More specific comments are below: 

Fig 1C, disorder/beta sheet/alpha helical labels are difficult to distinguish, using a solid line ? 

Fig. 2b-2d, the authors need more repeats to generate statistics of the Kd values to draw 
conclusion about whether the different mutants indeed bind auxin with different affinity. This kind 
of measurement usually has a high variations. Also again, the curves are difficult to distinguish, 
please use different colors. 

Supplementary Fig 1: would be nice to label the position of the degron in each panel. 

Fig. 1C, since IAA7 and IAA17 are mostly disordered, one would expect that it would be hard to 
purify the proteins. A coomassie staining gel of the purified proteins would be required to show the 
purity/integrity of the proteins. In addition, since the proteins are purified from E. coli, one cannot 
rule out that the prokaryote system might not fold the protein properly. Have the authors tried 
AUX/IAA expression in insect cells as TIR1-ASK1 ? 

Page 11 line 191, there is an extra “12” that needs to be removed. 

Page 12 line 205, Should be “Supplementary Fig. 4d”. 

Page 12 line 215 Should be “Taken together” 

Supplementary Fig 8b, are these quantifications from several independent experiments, could the 
authors put p-values on the figure to indicate statistical significance. This is even more relevant 
given that in Figure 3, it really difficult to see the difference of the fluorescence intensity just by 
eyes, for example between 77777 and 777127. Also the orders of the samples between 
Supplementary Fig 8 and Figure 3 are different. Please make sure about the labeling and stick to 
the same order. 

Fig. 5a-b, What are the restrains indicated in the x-axis ? Why is the additional degron tail 
restraint only restricted the accessible conformations in the last column group ? 

Reviewer #2 (Remarks to the Author): 

Auxin is the most critical hormone in plant biology, driving all cell division and cell elongation 
events. Despite its great importance, its signal transduction pathway is seemingly simple, with a 
repression – depression paradigm, driven by auxin-mediated interactions between the SCFTIR1 
complex and an Aux/IAA repressor, ruling auxin responses. 

In this manuscript, the authors use a variety of approaches to resolve the topology of TIR1 contact 
points with IAA7 and IAA12, with a focus the C-terminal intrinsically disordered regions of these 
proteins. 

Overall, this work will provide important details to the auxin community that may also be relevant 
to interactions among other F-boxes with their targets. 

I have a few minor comments for improving this manuscript. 

1- Line 264– It seems strange to me that the authors did not cite Gilkerson (2015) who showed by 
mutational analysis that Aux/IAA polyubiquitylation is flexible and can take place on many 
potential residues. At the very least, this should be discussed in the context of their work. 

2- Line 346 – The authors make a strong statement about the numbers of assemblies detected for 
IAA12bm3 and IAA7bm3 and distribution of inter-protein crosslinks. Based on difficulties in 
identifying all peptides in mass spec, I am not comfortable with such a strong conclusion – the 
authors should soften their statements on this. 

3- The theme of hugging and kissing and embracing throughout the discussion to describe TIR-
Aux/IAA interactions seems inappropriate. I am guessing that a “hug” and a “kiss” are meant to 
depict different types of interactions, but it is difficult to tell. I would suggest using more specific, 
non-colloquial, language. 

Reviewer #3 (Remarks to the Author): 

Authors used interdisciplinary approach and combined an in silico analysis, biochemical analysis of 
recombinantly expressed proteins, the yeast two hybrid system (Y2H) for qualitative assessment 
of auxin co-receptor assembly as well as ubiquitylation dynamics of selected proteins (IAA7 and 
IAA12). Using spectroscopic (CD) and diffusional experiments in combination with recombinantly 
expressed proteins they found that N terminal part of these proteins is disordered with an 
extended structure in solution. 
Using Y2H approach of chimeric proteins they investigated impact of intrinsic disordered segments 
as well as of the compact PB1 domain of AUX/IAA (IAA7 and IAA12) proteins on their interaction 
with TIR1, auxin independent, as well as auxin-triggered. 

They stated that they confirmed the postulated interdependency of the degron tail and PB1 domain, 
but I am not convinced since chimeric proteins without PB1 nicely bind to TIR1, also, there in no 
experimental data (or I overlooked them?) on the PB1 - TIR1 binding. They point to additive and 
separate effects of each, disordered degron tail and the PB1 domain, on auxin independent and 
auxin-triggered TIR1 interaction. Furthermore, they postulated that AUX/IAA ubiquitylation 
favorably occurs at the solvent exposed regions in IAA7 and IAA12, when they are recruited by 
TIR1. Many assumption but without firm evidences. 

Based on the structural proteomics approach authors assumed AUX/IAAs IAA7 and IAA12 exhibit 
flexible conformations in solution (intra-protein cross-links), and adopt an extended fold when 

bound to TIR1. But the cross-linking reactions were performed for chimeric (PB1 missing domain) 
proteins, and to get more convincing assumption the data for the native proteins are needed. 

Y2H 
I am not an expert, but from the data shown, it is hard to follow the statements given in the 
manuscript. For example: 
1. The statement on IAA7 and IAA12 – TIR1 binding affinity ' Expressionof the ß-galactosidase 
reporter indicates stronger interaction of TIR1•IAA7 than TIR1•IAA12.' Is not correlated with Fig. 
2a, since according to the data shown it seems that TIR1•IAA12 bind only at VERY high IAA conc. 
(> 100 µ 
M). 
2. Author wrote that they docked homology-modeled PB1 domains of Arabidopsis IAA7 and IAA12 
to the ASK1∙TIR1 complex, applying distance restraints based on the cross-linking data. The 
mentioned distance restraints are related to the regions out of PB1, and it is not clear how authors 
modeled 3D structure of these regions? 

Ubiquitylation 

3. The statement: 'IAA12~ubiquitin conjugates were much less abundant than IAA7 after 30 min 
incubation' can not be derived from the data represented in Fig3a. From Fig. 3a it seems that they 
(IAA7 and IAA12) are similarly ubiquitinated. 

Cross-linking 
How long is the cross linker used? Are there data on its influence on the protein conformation? 

Figure 6: 

4. Fgs 6a and 6b - From the Δ graphs it seems that the affinity of PB1(IAA7) for TIR1 is lower 

than that of PB1(IAA12) – TIR1, while according to the experiments IAA7 binds to TIR1 with higer 
affinity than IAA12 
5. The following statement: 'Mutations R156E, as well as S201A, and S205A either abolished or 
drastically impaired basal TIR1•IAA7 and auxin-driven TIR1•IAA7 and TIR1•IAA12 associations, 
without affecting ASK1•TIR1 assembly.' could not be deduced from Fig 6 e. According to the figure 
the statement is only partly true for R156E mutants of IAA7 at low auxin conc. (0.1 and 1 uM), but 
for IAA12, as well as for IAA7 at higher IAA conc, there is no significant difference with respect to 
the WT proteins.6. 

Fig2b, the affinity curve for IAA/bm3 have not reached the saturation (plateau) 

The computational methods used in this manuscript is really strange: 
1. Authors performed homology modelling of IAA7 and IAA12 PB1 domain, but what is with the 
other regions of the protein (DI, linker, degron tail?). How many and what structures were used as 
templates? What was the quality of the obtained models, how big is the 3D structure? 

2. Authors wrote that 'how to incorporate distance restraint' as input for HADDOC can be found 
elsewhere (they did not give a reference), but these restraints are related to regions out of PB1 
while they have homology model only of PB1 domain. How they combined distance restraint data 
with non existing 3D structure for the purpose of docking? 

3. Molecular modelling procedure: Structures (PB1(IAA7)-TIR and PB18IAA12)-TIR) were 
optimized in MOE (using AMBER10 ff), 
But there is no reference. As far as I know AMBER10 ff does not exist! Futher on, the simulations 
were performed in GROMACS with AMBER99SB-ILDN force-field (the energy minimization and MD 
simulations should be performed with the same ff). Authors states that 'electro-neutrality was 
guaranteed with a NaCl concentrationof 0.2 mol/L'. What was net charge of the system! Electro-

neutrality can not be achieved by adding the same amount of Na+ and Cl-, but, by adding certain 
number of either Na+, or Cl- ions, depending on the charge of the system. 
Nothing about time step, ensemble, barostat, thermostat ... 
Finally, the binding free energies were calculated by MMPBSA.py from Amber18 package. Why, not 
in Gromacs, since it was used for MD simulations, and it has his own MMPBSA tool. Why usage of 
so many different program packages (and ff), when everything could be done within either 
GROMACS the ABER18 package? 

Figures 6 a & b: Authors wrote: 'The accumulated mean value of Δ were obtained every 10 ps' 
What does mean 'accumulated'? It should be the ' mean value of Δ' (or running average). 

What was the frequency of sampling? 

In general the sentences are too long, awkward and imprecise, for example: 
' The residency time of an AUX/IAA target on TIR1, when assembled in an SCF-complex, propels 
processivity of AUX/IAA ubiquitylatio...' 
(How time can propels something, maybe correlate OR define ...) 

' IDRs flanking the degron provide high flexibility and an extended fold to AUX/IAAs' 
Should be 'ensure' (or enable..) 

'ΔGeff values over 20 ns identifying and refining stable TIR1•AUX/IAA PB1 complexes'... 
Identifying is OK, but the calculated ΔGeff values do not refine structures! 

Also, some citations are (in my opinion) not adequate, for example for the statement 'Under low 
auxin concentrations, AUX/IAAs repress type A AUXIN 53RESPONSE FACTORS (ARF) transcription 
factors via physical heterotypic interactions through their type I/II Phox/Bem1p (PB1) domain' 

authors cite: Liscum, E. & Reed, J. Plant Mol Biol (2002) 49: 387, 
instead of 
Tiwari S.B., Hagen G., Guilfoyle T.J. (2004). Plant Cell 16: 533–543 
OR 
Tom J. Guilfoyle Plant Cell. 2015 Jan; 27(1): 33–43. 

Reviewer #4 (Remarks to the Author): 

The paper by Niemeyer et al presents an interesting hypothesis on the role of intrinsically 
disordered regions in the auxin-mediated interaction between INDOLE-3-ACETIC ACID proteins 
(transcriptional repressors) and F-BOX PROTEINS TRANSPORT 57 INHIBITOR RESPONSE 1 
(TIR1)/AUXIN SIGNALING F-BOX 1-5 (AFB1-5). The authors claim as their main conclusion that 
the intrinsic flexibility of AUX/IAAs ultimately affects the ubiquitination system, and in turn 
proteasome recruitment. With this angle, we believe the manuscript has considerable impact due 
to the crucial role of the ubiquitin-proteasome system which, in combination with the effect of 
Auxin concentration, is crucial to regulating plant growth. 
The work represents additionally a serious effort in its experimental setup, in which the orthogonal 
approach combines the generation of many seamless chimeric IAA7 and IAA12 complexes with 
binding assays, MS-based proteomics, CD spectroscopy and in-silico protein docking and molecular 
dynamics simulation. The biochemical work appears to be high class work. However, we found 
some rather speculative conclusions from a structural point of view and a number of errors & 
oversights in the experimental procedures which potentially lead to inaccuracies in the study. 
Mindful of the impact of the story, we recommend major revisions and detail the specific points 
below in hopes of providing constructive points aiming to improve the manuscript and making it 
suitable for publication. 

Major points 
[1] In general the manuscript is not easy to read for readers lacking a solid background on the 
topic. We feel this is important given that the scope of the journal is to address a wide audience. 
To improve this, the abstract and introduction should be more general and with the basic concepts 
better explained without the need of reading all references cited. A major point here is to use less 
acronyms, which makes the text very difficult to follow. Additionally, the authors should put efforts 
in explaining why this work, which is primarily important for plant biologists, is also important to a 
more general audience. This can potentially be achieved by highlighting which technical/biological 
novelties are presented. 

[2] Inspection of the repository containing the raw data of the cross-linking experiments appears 
to indicate a peculiar weakness in the data analysis of the raw data. The repository provides a 
FASTA file containing only the 4 proteins in the complex. If really used, this is directly counter to 
the recommendations of the Sinz laboratory, who have promoted the use of cleavable cross-linking 
reagents for many years to support the analysis of raw data with large(r) FASTA files. In addition, 
such low numbers of proteins will make FDR controlling the identifications at the indicated level 
impossible – although this situation will at least partly be rescued by only selecting reproducible 
cross-links. The authors should re-analyze the data with larger FASTA files to make the data 
analysis more secure. 

[3] The number of cross-linked peptides seems very low as IAA7 and IAA12 targets contain many 
LYS residues and respectively 3 and 6 were found ubiquitinilated. Such low density is counter-
productive to the structural modeling steps the authors apply based on this data. To capture a 
higher density of cross-linked peptides, the authors can consider to employ pre-fractionation for 
their cross-linking reagent of choice for the samples after digestion to enrich for the cross-linked 
peptides and measure multiple fractions (as opposed to the single ‘shot’ currently analyzed). This 
obviously will depend on the amount of input material the authors will be able to generate, but will 
greatly assist in the modelling steps and increase the confidence in the results. 

[4] Line 719: it is not clear why the authors choose to use cross-links present in 2/3 or 3/4 
replicates and not to be consistent in this parameter – please comment on this. Additionally, it is 
currently impossible to assess which crosslinks were present in which replicate as the excel file in 
the PRIDE repository is corrupt (likely a result of a mistake in the data transfer). To improve on 
this point, apart from the need of updating it in the PRIDE repository to a non-corrupt version, the 
complete table containing the information and sequences of the crosslinks in the various replicates 
should be included in the supplementary material. 

[5] Line 713: the cross-linkable residues set up in the search included LYS, SER, THR and TYR. To 
us, it is not clear how reliable these hits are, given that the preferable residues should be LYS-LYS, 
and what inclusion does to the false positive rates (a point for example apparently not researched 
in the recent paper Beveridge et al currently available in bioRxiv). The authors should reanalyze 
the data with LYS-LYS only and comment on the effect. Additionally, as the table containing the 
identifications in the PRIDE repository appears to be corrupt it is currently impossible for us to 
estimate the validity of the results (see also point 4). 

[6] While inspecting the raw MS data for the XL-MS experiment it became clear that different 
instrument and acquisition settings were used. For instance, at least one of the samples (IAA12 
rep.1, minIAA, plusT1) was acquired on a Q Exactive Plus with an MS2 resolution of 70’000 with 
stepped collision energy, while replicate 4 of the same sample was acquired on an Orbitrap Fusion 
with an MS2 resolution of 15’000 without stepped HCD collision energy. The above mentioned 
parameters are different from the one stated in the methods section of the manuscript. The 
authors should provide the correct settings in the manuscript and provide a rationale for the 
differences and/or potential effect. 

[7] Line 728: “… we used distances from intramolecular cross-links of known distance (see SUPP 
file docking parameters)”. The HADDOCK parameters are in fact not provided and it is also not 
clear which cross-links were used in which step, making it impossible to reproduce the result 
(Supplementary Table 3 contains solely information about the models generated by HADDOCK). 
Additionally, with “known distances” do the authors refer to the spacer arm length of the linker or 
the length of the side-chains added to the spacer arm length? In extension to this, are for example 
the detected Tyr then also adjusted with a different side-chain length? Many open questions which 
definitely can and should be answered by including a table with these details. 

[8] Line 735: “Using the same restraints, the possible conformational docking space of the PB1 
domains was searched and visualized using DisVis…”. As stated by the authors who developed 
DisVis (ref 44 of this manuscript, doi: 10.1093/bioinformatics/btv333 ), DisVis is a “…. powerful 
aid in detecting the presence of false-positive restraints”. This means the tool should be used prior 
to the docking steps to select the set of distance restraints that define a specific interaction 
interface for optimal performance (also recommended in all their workshops). Supplementary 
Figure 11 appears to indicate the opposite in that both tools were run in parallel. In case this is 
true, the authors should re-run the docking procedures informed by DisVis. See point 7 on how to 
provide further details. 

[9] Line 333: “The location of the clusters on two opposing surfaces of TIR1 334 suggests a rather 
extended fold of the AUX/IAA protein when bound to TIR1”. How do the authors infer this 
extended fold of the protein if most of the crosslinks link the N-terminus to the C-terminus (see 
circus plots in Figure 4c and d)? We are wondering if cross-linking the recombinant expressed 
proteins with varying disordered tails would not be an excellent system to demonstrate whether 
the terminal binding remains intact – and if not whether this is the reason behind decreased 
binding. 

[10] In extension to point 9, it seems strange that no extensive cross-linking is found on or in the 
neighborhood of the degron binding domain as this appears to be the main interaction point and 
the most stable point between the two proteins – which should lead to availability of many cross-
linking options. The authors should comment on likely reasons to this, for example are no LYS-LYS 
pairs available in the interfaces between the proteins in the final model or other reasons. 

[11] Figure 7: It is not clear how the authors came to this model. The PB1 domain, according to 
the reproducible cross-links considered, is linked to the K226 in the position “cluster 1” for the 
IAA7 (Figure 4c), while the KR motif at the N-terminus is linked to the K67 (not defined as a 
cluster but close to the ASK1 domain (Supplementary Fig. 9). On the contrary, the PB1 domain of 
IAA12 is linked to K67 which is not compatible with the proposed model. Do the authors expect 
different sites of interaction for the PB1 domains of IAA7 with respect to IAA12? 

Minor points 
[12] Line 726: “…description how to prepare pdb files and incorporated distance restraint can be 
found elsewhere.” Please provide a reference. 

[13] Line 722: The comparative models of IAA7 and IAA12 PB1 Domains were created, but the 
PDB files are not available. Their inclusion we feel is important to allow readers to assess the 
claimed structural interactions based on the crosslinks found. Please provide these PDBs and 
possibly also the starting models used for the molecular dynamics simulation. 

[14] Figure 5: The title specifies: “.. Cross-link-based docking..” and “… via HADDOCK…”. The 
structures presented however are not docked PDBs, which is an HADDOCK output, but rather the 
possible conformations space occupied by the PB1 domain, which is an output of DisVis. Please 
here provide the proper docked structures or correct the figure caption. 

[15] Line 327: “… in the absence of auxin, we only observed only a few inter-protein and similar 
intra-protein cross-links…”. It is unclear to us from the provided figures 4 and sup 10 that this is 
the case. The authors should clarify this in more detail what the difference is.