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



Reviewers' comments: 

Reviewer #4 (Remarks to the Author): 

The authors have performed an impressive array of work for this rebuttal and I commend them on 
their efforts. The readability of the manuscript has improved significantly and most of the requests 
for clarification about the structural modelling addressed. In addition, the MS experiment are now 
replicated and the instrumentation used is now consistent across the different runs. 

In my opinion there remain however three small points related to the modelling procedures that 
need to be addressed before publication, as I believe they are important to the work. 

[1] In the file “haddock_files.tar\haddock_files\IAA07\No_disorder_restraint\”, the PDB cluster 1_1 
is missing. In the summary of the run it is indicated as present. It is an important point to clarify 
as this cluster is the most significant (124 of the 175 structures) and the first structure is usually 
the highest scoring one, so it should be included or its absence explained. 

[2] The authors did a great effort to make the methods section detailed and reproducible. The 
summary file (e.g. : 05_multi-IAA12_COM2.docx) is however not provided for all HADDOCK runs. 
This file is essential to assess how many structures are present in the cluster, since a low amount 
of structures in a defined cluster might drive to misinterpretation in the following steps. 

In Figure 6, the legend (line 1180) states: “Time evolution of instantaneous ΔGeff values over 20 
ns identifying stable TIR1·AUX/IAAPB1 complexes from HADDOCK best scoring groups.”. And 
related to the same figure in the main text (line 375): “We used as a starting structure (t=0) the 
results from the HADDOCK simulations including the degron tail restraint.”. As all models are now 
provided, the best scoring groups from HADDOCK should be mentioned in a way they can be 
retrieved in the supplementary data. 

[3] DisVis-IAA07 (all-restraints) run was performed with only 4 restraints, one of which with a 
different minimum-maximum distance of 20 Å and 27.4 Å, respectively: 
A 226 CA B 228 CA 8.0 34.0 
A 229 CA B 228 CA 8.0 34.0 
A 2013 CA B 124 CA 20.0 27.4 
A 2013 CA B 242 CA 8.0 34.0 

Same for the DisVis-IAA07 (all-restraints) run, an apparently arbitrary range of minimum-
maximum distance seem to have been set (24.8 Å and 36.0 Å, respectively): 
A 226 CA B 233 CA 8.0 34.0 
A 67 CA B 233 CA 8.0 34.0 
A 2013 CA B 121 CA 24.8 36.0 

These distance ranges are narrower than the distance restraint set for the others, and from what is 
expected for the DSBU cross-linker itself (which should be 34 Å, as stated by the authors). 
The authors should clarify in the method section why a subset of restraints was used to run DisVis 
analysis, and how these have been selected since they are different from those provided in the 
Supplementary Tables 2 and 3. 

Reviewer #5 (Remarks to the Author): 

According to my opinion, the authors answered to the most of the remarks made by Reviewer#3. 
It is very good that the authors provided detailed description of the computational methods in the 
Supplement Info. However, I fully agree with the Reviewer#3 that a sentence or two regarding the 

details of the MD simulations including thermostat, barostat, time step and ensemble should be 
added in the Methods part of the manuscript. Also, I am deeply concerned about the short 
simulation time of only 20 ns. At least RMSD graphs of the proteins' backbone during the MD 
simulations must be provided, either to convince the reader that the simulations were equilibrated, 
or to give a chance to the reader to judge by itself how equilibrated the systems are and how 
reliable are the computational results due to short simulation time. Altogether, it is an interested 
paper, but before publishing, two issues regarding the computational part should be done: 1) to 
add a few details regarding the computational procedure and 2) to add RMSD graphs of the 
simulations in manuscript.