Insights into VDAC Gating: Room-Temperature X-ray Crystal Structure of mVDAC-1

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Kristofer et al. showed a structure of mouse VDAC-1 revealed by room-temperature X-ray crystallography, which displays notable differences from the previously determined structure solved in a cryogenic condition, suggesting a functional transition of VDAC. By applying Electric Field-Stimulated X-ray crystallography, the authors induced voltage-driven conformational changes to resolve the ‘closed’ state of VDAC-1. This technical approach can surmount the previous difficulties in studying voltage-dependent conformational changes of the ion channels. 

 

Overall, this is an interesting work and can benefit the field of studying VDAC and related voltage-dependent anion channels. I do not have a major concern in this study but have some comments and questions that need to be addressed. 

 

1.      The authors used a PDB coordinate, 3EMN, as a ‘cryogenic’ X-ray structure to compare with their ‘room-temperature’ structure. However, it is unclear whether 3EMN is a cryogenic structure because the experimental information section on the PDB website says that the X-ray data of 3EMN was collected at 298K (~25°C), while there is no other information described in the original literature. Can the authors address how they confirmed that 3EMN is indeed a cryogenic structure?

 

2.      Lines 177 to 182, the authors say that “…the beta barrel exhibits small deformations when comparing RT and cryo conditions, likely caused by the anisotropic contraction of the unit cell during freezing…” and also “The observed conformational differences, although minor, may have implications in transitioning between the ‘open’ and ‘closed ‘state of VDAC-1”. These statements are confusing. Is it saying that the channel of the cryogenic structure is actually wider than the RT structure, despite the contraction? This needs to be clearly explained, as it seems counterintuitive.

 

3.      Related to the question above, can authors show a cross-section view or interior surface of the channel in different temperature conditions and compare the inner sizes of the channel to show the functional state of the RT VDAC?

 

4.      Lines through 204 to 209, regarding the high B-factor regions, can the authors compare the crystal lattice of the cryo and RT structures to see if there is any interaction that influences these regions, which potentially causes the B-factor differences between the two structures?

 

5.      Line 214, with the current data, there is no strong connection between the high B-factor regions and their implication of the open and closed states transition. The authors may need additional experiments, such as mutational assays, to support this idea.

 

6.      How can the authors reconcile their observation with the previously known regulatory mechanism by the central helix inside the channel as they are almost the same between the cryo and RT structures?

 

7.      Many figures are demonstrated in very similar ways but not properly show the contents in the text. For example, in lines 168 to 172, there are many residue level descriptions in the text but their labels and detailed presentations are missing in the relevant figure. I recommend the authors show them in more appropriate ways.

 

Minors

1.      Figure1, please describe the full name of ‘IMS’. This seems to appear later in the manuscript, though, it is more appropriate to be addressed here.

 

2.      Line 92, ‘a-helical’ should mean ‘a-helical’.

 

3.      Lines 174 and 177, say Figure 5A and Figure 5B, but there are no such panels in Figure 5.

 

4.      Figure 6A, both b2 and b6 strands are colored gray, and they are not distinguishable in the figure. Please label them for clarification.

 

Author Response

Reviewer 1:

Overall, this is an interesting work and can benefit the field of studying VDAC and related voltage-dependent anion channels. I do not have a major concern in this study but have some comments and questions that need to be addressed. 

Thank you for your thoughtful critique and for recognizing the potential impact of our work on the field of VDAC and voltage-dependent anion channels. We appreciate your valuable feedback, and we have carefully addressed your comments and questions. Your insights have helped strengthen the manuscript, and we believe the revisions have resulted in a more robust and comprehensive paper.

 

We have outlined below how we have incorporated your suggestions into the revised manuscript.

 

1. The authors used a PDB coordinate, 3EMN, as a ‘cryogenic’ X-ray structure to compare with their ‘room-temperature’ structure. However, it is unclear whether 3EMN is a cryogenic structure because the experimental information section on the PDB website says that the X-ray data of 3EMN was collected at 298K (~25°C), while there is no other information described in the original literature. Can the authors address how they confirmed that 3EMN is indeed a cryogenic structure?

 

Thank you for catching this oversight, and I appreciate your careful review of the details! I can confirm that 3EMN was indeed collected at cryogenic temperatures, as I was present for the data collection and am the corresponding author on the original paper. The entry on the PDB website incorrectly lists the data collection temperature as 298K, which is an error. I will contact the PDB to have this corrected.

 

2. Lines 177 to 182, the authors say that “…the beta barrel exhibits small deformations when comparing RT and cryo conditions, likely caused by the anisotropic contraction of the unit cell during freezing…” and also “The observed conformational differences, although minor, may have implications in transitioning between the ‘open’ and ‘closed ‘state of VDAC-1”. These statements are confusing. Is it saying that the channel of the cryogenic structure is actually wider than the RT structure, despite the contraction? This needs to be clearly explained, as it seems counterintuitive.

 

Thank you for pointing out the confusion in our statements. We agree that the explanation was unclear and could lead to misunderstandings. We have now revised the text to clarify that the β-barrel shows a small reduction in volume when comparing the room-temperature (9,102 ų) and cryogenic (10,314 ų) structures. We believe this reduction indicates that the β-barrel could transitions to a more compact form, which could potentially relate to the barrel's ability to undergo conformational changes to the closed state. We hope this modification makes the comparison more intuitive and better supports the discussion of the ‘open’ and ‘closed’ states of VDAC-1.

 

3. Related to the question above, can authors show a cross-section view or interior surface of the channel in different temperature conditions and compare the inner sizes of the channel to show the functional state of the RT VDAC?

 

Thank you for the suggestion. We have now added a new figure (Figure 6) that illustrates the slight deformation of the β-barrel under different temperature conditions. This figure compares changes in the walls of the β-barrel between room-temperature and cryogenic conditions, providing a clearer visualization of the structural changes. We suggest this deformation as a possible mechanism for contracting the barrel during the transition to the closed state.

 

 

4. Lines through 204 to 209, regarding the high B-factor regions, can the authors compare the crystal lattice of the cryo and RT structures to see if there is any interaction that influences these regions, which potentially causes the B-factor differences between the two structures?

 

We examined the regions with higher B-factors as shown in Figure 7. The loops between β-strands 1-2, 5-6, 8-9, and 18-19 are not involved in crystal contacts. However, a region near the β-strands 18-19 loop and β-strands 2 and 3 is involved in a symmetric interface of the unit cell. Based on this analysis, it appears that the crystal contacts are unlikely to be the cause of the B-factor differences between the cryo and RT structures.

 

6. How can the authors reconcile their observation with the previously known regulatory mechanism by the central helix inside the channel as they are almost the same between the cryo and RT structures?

 

The regulatory mechanism of the central helix inside the channel is still very much debated, which is precisely why obtaining a structure under applied voltage is crucial for advancing our understanding. Early models suggested that the central helix would exit the pore entirely during gating, but this mechanism has been debunked by several studies. More recent publications sugest that the gating mechanism may involve a rearrangement of side chains and partial pore collapse, leading to the closed state. This hypothesis is consistent with our observations, as the structural differences between the cryo and RT structures may reflect the initial steps in this conformational shift.

 

 

7. Many figures are demonstrated in very similar ways but not properly show the contents in the text. For example, in lines 168 to 172, there are many residue level descriptions in the text but their labels and detailed presentations are missing in the relevant figure. I recommend the authors show them in more appropriate ways.

 

Thank you for pointing this out. We have now revised the figures to ensure that all relevant 'stick' residues mentioned in the text are labeled accordingly. This should make the figures more informative and aligned with the residue-level descriptions provided in the manuscript.

 

Minors

All minor errors listed below have been corrected.

1. Figure1, please describe the full name of ‘IMS’. This seems to appear later in the manuscript, though, it is more appropriate to be addressed here.

 

2. Line 92, ‘a-helical’ should mean ‘a-helical’.

 

3. Lines 174 and 177, say Figure 5A and Figure 5B, but there are no such panels in Figure

 

4. Figure 6A, both b2 and b6 strands are colored gray, and they are not distinguishable in the figure. Please label them for clarification.

 

 

Author Response File: Author Response.docx

Reviewer 2 Report

Comments and Suggestions for Authors

The paper presents a room temperature structure of the mitochondrial outer membrane protein VDAC1. The protein is a major player in the function of the mitochondria with functional aspects inevitably touching a variety of biological processes given the importance of the organelle.

The closed states of VDACs are indeed unknown and a highly desired target.

The strengths of the article are as the authors described: This is a new structure of VDAC1 and the path to the closed state is feasible.

The major weaknesses of the article are obvious and clearly stated by the authors. The deviations from the cryogenic structure are minimal and the solution of the structure in an electric field is neither guaranteed nor guaranteed to give a closed state.

Given the importance and relative novelty I strongly recommend publication.

If the authors are able to list functionally relevant mutations in the vicinity of the structural and B-factor differences it would perhaps motivate their hypothesizing more strongly that this structure is relevant to the closed state. But I do not recommend expanding the writing extensively as it is concise and clear in its present form.

 

 

 

Author Response

Reviewer 2:

Given the importance and relative novelty I strongly recommend publication.

Comment:

If the authors are able to list functionally relevant mutations in the vicinity of the structural and B-factor differences it would perhaps motivate their hypothesizing more strongly that this structure is relevant to the closed state. But I do not recommend expanding the writing extensively as it is concise and clear in its present form.

 

Thank you for this suggestion. While many mutations have been studied in VDAC, very few point mutations significantly alter gating. The most prominent mutation is on the helix at K12, which we have highlighted in the text. However, this region does not coincide with areas that show significant changes in B-factors.