Peer review of: A distal regulatory region of a class I human histone deacetylase (2020)

REVIEWER COMMENTS 

Reviewer #1 (Remarks to the Author): 

The manuscript titled “A Distal Regulatory Region of a Class I Human Histone Deacetylase” 
addresses importance of allosteric effect of two distant -helices (H1 and H2) on the active site 
conformation of HDAC8 enzyme. 
The authors made an excellent study showing the allosteric property in detailed utilizing all 
possible experimental and computational methods. Although recent and older work exist in the 
field, not many scientists brought the hypotheses and data together to make a picture out of 
accumulative knowledge of the allosteric effect of HDAC Class I enzymes so far. 
It is original, nice flow and the title clearly reflects its contents. It is well organized. I believe the 
manuscript is timely and will get the attention of many researchers studying in this field. The 
paper is publishable in the present form with the following comments: 

Comment: 

1. Why is it needed to run 4 micro second simulation? Is the system reaching to equilibrium after 
such a long time? No explanations are given on this. 

2. It would be nicer to see the progress of MD simulations of substrate bound HDAC and TSA 
bound HDAC enzymes. 

For example; Radius of gyration, time versus RMSD graph of equilibrated and crystal structure 
enzymes and time versus RMSF of substrate bound and TSA bound enzymes especially in the H1 
and H2 helices. It would be easier to follow these fluctuations on the graphs over the simulation 
time. 



Reviewer #2 (Remarks to the Author): 

In this manuscript, Werbeck et al. describe solution NMR studies exploring intrinsic dynamics of a 
human histone deacetylase, HDAC8, and show that a lowly populated species of the wild-type 
protein represents an inactive state of the enzyme – a state that is selected by a common HDAC 
inhibitor. The results described are of great interest not only to investigators in the field but also to 
the broader scientific community including enzymologists, NMR spectroscopists, theoretical 
biophysicists, among others. 

The authors use a combination of spin relaxation experiments, mutagenesis, and molecular 
dynamics simulations to identify an allosteric mechanism of enzyme regulation that has 
implications for other Class I HDACs because the same region identified in HDAC8 was previously 
implicated in the positive (allosteric) regulation of enzyme activity by small molecules and 
associated subunits. However, crystal structures of these HDACs could not explain the mechanism 
of allostery and it was suspected that the inactive conformation was poorly populated. In a sense, 
this question could only be answered through studies of HDAC8 as this is the only HDAC that could 
be expressed in its properly-folded form in E.coli and studied by NMR. 

I do have some concerns, but they could be easily addressed through additional experiments or 
appropriate revisions to the text. Some of these are because the authors are either unfamiliar with 
the literature or did not describe previous findings accurately. 

1. Line 35 … HDAC1 and HDAC2 show abundant deacetylase activity; it's HDAC3 that is inactive in 
the absence of the SMRT DAD domain (see Schultz et al., Biochemistry 2004, 43, 34, 11083-

11091) 

2. Line 36 … The activity of HDAC1 and HDAC2 is enhanced several-fold by inositol phosphates and 
other HDAC complex subunits such as MTA1 (see Millard et al., Mol Cell 2013, 51, 57-67) SAP30 
and RBBP7 (see Marcum & Radhakrishnan, JBC 2019, 294, 38, 13928-13938). 

3. Line 37 … HDAC8 is not an "isoform"; it could, however, be called an isozyme. 

4. Line 37 … HDAC8 is the least active HDAC (see Schultz et al., (2004) above) but it shows 
enhanced activity in the presence of metals such as Fe(II) and Co(II) than Zn(II) (see Castaneda 
et al., Biochemistry 2017, 56, 42, 5663-5670). But even then, its kcat/KM is orders of magnitude 
lower than the other Class I HDACs. 

5. Line 42 … Define "active apo form" 

6. Lin 84-88 … The original structure of HDAC8 in complex with TSA featured 2 TSA molecules 
(PDB ID: 1T64); could the shifting of resonances observed in Fig. 1c during the latter half of the 
titration be due to binding to a second equivalent of TSA with a lower affinity for the interaction? 

7. Line 162 … How was the enzymatic activity data measured and what does %age activity mean 
for the various HDAC8 mutants? 

8. Line 191-195 … While this would otherwise be a reasonable approach, given that the peaks 
even for the annotated isoleucines do not fall along the vector connecting the apo and inhibitor-
bound states, it is recommended that independent spin-relaxation measurements be performed for 
extra validation. 

9. Figure 4: From the MD simulations, is it obvious what causes the loop conformation to change 
its bias towards the inactive conformation; in other words, does the side chain of E39 have a role 
in this process? If so, what is it? 

10. Figure 4a/e: It would be useful to compare the distance distributions from the MD simulations 
for TSA-loaded WT with those for the apo WT and S39E mutant. 



Reviewer #3 (Remarks to the Author): 

Werbeck et al present the study on the conformational flexibility of HDAC8. The authors show 
clearly that HDAC8 is allosterically regulated by shifts in populations between exchanging states. 
The manuscript is exciting and the study was performed thoroughly. The major result is originated 
from the fact that chemical shifts acquired from relaxation dispersion of wild type match with 
chemical shift difference between wild type and TSA-bound forms. The work was carefully 
conducted and drawn conclusions are appropriate and reasonable. The reviewer finds only a few 
minor issues as follows: 
a. the authors present only chemical shift difference between TSA-bound form and wild type 
(Figure 2 d). How does the correlation between chemical shifts obtained from relaxation dispersion 
and chemical shift difference between other compounds and wild type? 

b. only carbon chemical shifts have been used for the correlations between chemical shifts 
obtained from relaxation dispersion and methyl-trosy (Figure 2 d). How does it look with proton 
chemical shifts? 

c. although the reviewer believes that Figure 3c has been obtained based on populations from 
slopes between chemical shift variances from relaxation dispersion and chemical shifts from 
methyl-trosy (Figure S7). But there is no description. The reviewer thinks that the populations 
obtained from carbon chemical shifts. How is the correlation using proton chemical shits? 

d. Molecular dynamics provide interesting insights for structural changes. However, chemical shifts 
of methyl groups calculated with the structures from molecular dynamics would be better for direct 
comparison with experimental chemical shifts.