Peer review of: Human aminolevulinate synthase structure reveals a eukaryotic-specific autoinhibitory loop (2020)

Reviewers' comments: 

 

Reviewer #1 (Remarks to the Author): 

 

I am a computational chemist by training and have focused in my evaluation on the computational 
aspects. Nonetheless, I have some questions to the experimental parts as well. 

 

The manuscript describes the determination of a structure of an N-terminally truncated version of 
the human aminolevulinate synthase (hsALAS2). The C-terminal extension (Ct-extension) was found 
to be in a position where it has an autoinhibitory effect, which offers new explanations for observed 
genetic disorders. The authors study the dynamics of the Ct-extension using molecular dynamics 
simulations. Furthermore, they have performed kinetic studies on some mutants and performed a 
crystallographic screen for putative inhibitors. The work seems carefully done, is clearly described, 
describes novel findings and addresses a relevant topic. 

 

I would like to make the following comments: 

1. The authors perform simulations of three distinct states of the protein (and one fourth, with a 
putative inhibitor bound). In order to model the substrate bound state, they start from a 
representative structure of the most common cluster of the simulation with PLP bound, but without 
substrate. This means that in that simulation, the Ct-extension already moves away from the 
position that is observed in the X-ray structure. That is a discrepancy that needs to be discussed. 
Seemingly, the dynamics in the Ct-extension already take place when the substrates are not bound? 

2. The authors have modeled missing residues in the structure based on a homology model that was 
based on the scALAS structure. Does this also include the loop 549-555, which was not observed in 
the hsALAS2 structure? That would not make sense as figure 2c,d and the text in lines 210 – 212 
shows that these structures are very different in this region. As this flexible loop is very likely to 
affect the dynamics of the Ct-extension, the authors should describe in more detail how it was 
modeled. Could an inappropriate model of this loop lead to enhanced dynamics of the entire Ct-
extension and thereby lead to the movement of helix 15, already in the substrate free structure? See 
point 1. 

3. It is difficult to assess if the asymmetry between the monomers, which is described in the lower 
panels of supplementary figures 10 and 11 is a true observation, or if this is just luck. As the systems 
seem much more dynamic, two seemingly random trajectories may seem asymmetric. This can only 
be resolved by performing technical replicates of the simulations. 

4. The PLP in supplemental figure 7 seems different from the structure in figure 1e. The carbonyl 
oxygen is colored blue, and points in a very different direction in figure S7. 

5. Relevant details on the methodology of the molecular simulations are missing. How big was the 
simulation box, how were long-range electrostatics included, was the simulation performed at 
constant temperature and/or pressure, were bond-lengths constrained, what timestep is used? The 
Gromacs code is not listed under ‘software’ in the ‘reporting summary’. 

6. The identified fragments are tested for their inhibitory capacity by a single point experiment. For 
an inhibitor of this kind, it may be expected that full inhibition is not attainable. Determination of an 
IC50 would be appropriate to confirm a concentration dependent inhibition. 

7. More importantly, the inhibition of the WT is not the relevant measure here. Would the authors 
not much rather show inhibitory behavior of these compounds against delAT or Q548X ? 

8. Abbreviations ClpX and AAA+ are not explained. 

9. Homology is not quantifiable. Either two proteins are homologous (have a common ancestor) or 
they are not. In line 83, the authors describe a low-homology region. I guess they mean a region with 
low sequence similarity. 

10. Very few typo’s 

a. line 66: aldmine -> aldimine 

b. line 184: through pi-pi -> through pi-pi interactions 

c. line 319: two opening brackets 

 

 

 

Reviewer #2 (Remarks to the Author): 

 

This is an interesting paper that gives a bit more detail on the inhibitory action of the C-terminal 
region of aminolevulinate synthase. Alas2 has been recently shown to be a good target to control 
certain porphyrins through gene silencing approaches so there is potential therefore for small 
molecule drugs. 

 

The authors do repeat a lot of what is known already, but the paper does provide a structural 
interpretation of the C-terminal autoinhibition. The MD is pretty, but I am not sure it is that 
especially informative in this case. 

 

1. The key crystallographic point addressed here is the conformation of the C-terminal region 
because the story revolves around the structure of this autoinhibitory loop. I would therefore expect 

to see an omit map for this region to confirm its architecture and its correct placement in the 
electron density. 

 

2. I suspect the problem the authors have is that most of the C-terminal region is unstructured, 
flexible, or disordered. At least some of the C-terminal region should be well-structured to warrant 
the crystallographic build-up in the narrative. 

 

Provided there is good evidence for the C-terminal structure, then Nature Communications seems 
about the right level for this paper. 

 

 

 

 

 

Reviewer #3 (Remarks to the Author): 

 

A combination of crystallography, limited biochemical analysis of disease-causing mutations, and 
crystallography-based fragment screening were employed in this study on human ALAS2. MD 
simulations are included to support mechanistic inferences. The significance of the work is that the 
structural studies led to mapping of the C-terminal extension, providing a framework for formulating 
hypotheses about the molecular basis of disease-causing mutations for XLSA and XLP. Overall, the 
experiments seem to have been well conducted. However, the mechanistic insights to emerge from 
the study are limited. The fragment search led to the identification of ligands that were largely 
clustered in a hydrophobic pocket but had minimal inhibitory effects (~15% at 1 mM concentration). 
A key conclusion that the R511E mutation destabilizes the structure of the Ct-extension was not 
experimentally supported and other inferences of gain-of-function XLP mutation, tended to be 
overstated. The absence of quantitative data (kinetic data are shown without error bars, the number 
of replicates are not mentioned, values for kinetic parameters are not provided for wild-type versus 
mutant ALAS2) diminishes the study’s rigor. 

 

Major 

1. Line 126. The Km values for glycine and succinyl-CoA are not reported as stated in Fig S4a,b. These 
need to be noted with SDs in the text. 

2. Line 129. The data shown in Fig 4c does not establish interaction between ALAS2 and SUCLG1-
SUCLA2, but rather a messy gel with many bands. If the authors consider this conclusion regarding 
protein-protein interaction to be important for this study, then MS data establishing the identity of 
the proteins present in the putative crosslinked bands needs to be shown. 

3. Line 261/2: The statement that breaking the R511:E569 interaction renders the Ct-extension 
highly destabilized is not backed by experimental evidence. Furthermore, it is only marginally prone 
to proteolysis. Hence the conclusion that the R511 mutation is essential for maintaining the 
structural integrity of the Ct-extension is overstated. 

4. His-tags were retained during characterization of mutant proteins, which is problematic. The 
authors need to remove the Hi-tags to make meaningful inferences about the resulting disease 
phenotype. 

5. Line 272. The conclusion that the elongated C-terminal extension in the delAGTG variant is 
subjected to proteolysis is not experimentally supported. While a small amount of proteolysis is 
seen, its location is not mapped to the Ct-extension. 

6. Fig 4c,d. Error bars are not shown for the kinetic data. The number of replicates is not mentioned. 

7. Lines 279-286. The values of kcat and Km for wild-type versus each mutant that was 
characterized, need to be reported together with S.D. 

8. Line 283. Although it is stated that “we show here that delG has the same 2- to 3-fold wild-type 
efficiency (kcat/Km) with respect to succinyl-CoA as delAT and Q548X”, no such data are provided.   

9. Line 335. The statement, “a catalytically grounded state that prevents transition to the open 
conformation” is unclear. What is meant by catalytically grounded? And how can the authors rule 
out the much more like ensemble of conformational substates, with the equilibria being shifted by 
individual mutations? 

10. Line 342. What is the experimental basis of the conclusion that kinetically, “the Ct-extension 
exerts more influence towards product release rather than succinyl-CoA binding?” Km can not be 
equated with substrate affinity. 

 

Minor: 

1. Line 29. Change “That has evolved among higher eukaryotes” to “that is only present in higher 
eukaryotes”. 

2. Fig 1b. Monomer A described as being light green in the figure legend is shown in grey in the 
figure. Same problem in Fig. 1d, subdomain 2 and Fig 1e. 

3. Lines 90/91 (Supplemental section). Change “that could accommodate for the disordered” to 
“that could account for the disordered”. 

4. Line 184. Change “π−π” to “π−π interactions”. 

5. Line 191. FigS1 does not show the structure of succinyl-CoA bound ALAS2. Please correct. 

6. Line 365. Define FECH.