Dimerization and dynamics of angiotensin-I converting enzyme revealed by cryoEM and MD simulations
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eLife Assessment
This important study shows, for the first time, the structure and snapshots of the dynamics of the full-length soluble Angiotensin-I converting enzyme dimer. The combination of structural and computational approaches elucidates with convincing evidence the conformational dynamics of the complex and key regions mediating the conformational change. This work provides an example of how conformational heterogeneity can be used to gain insights into protein function.
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Abstract
Angiotensin-I converting enzyme (ACE) regulates the levels of disparate bioactive peptides, notably converting angiotensin-I to angiotensin-II and degrading amyloid beta. ACE is a heavily glycosylated dimer, containing 4 analogous catalytic sites, and exists in membrane bound and soluble (sACE) forms. ACE inhibition is a frontline, FDA-approved, therapy for cardiovascular diseases yet is associated with significant side effects, including higher rates of lung cancer. To date, structural studies have been confined to individual domains or partially denatured cryoEM structures. Here we report the cryoEM structure of the full-length, glycosylated, sACE dimer. We resolved four structural states at 2.99 to 3.65 Å resolution which are primarily differentiated by varying degrees of solvent accessibility to the active sites and reveal the full dimerization interface. We also employed all-atom molecular dynamics (MD) simulations and heterogeneity analysis in cryoSPARC, cryoDRGN, and RECOVAR to elucidate the conformational dynamics of sACE and identify key regions mediating conformational change. We identify differences in the mechanisms governing the conformational dynamics of individual domains that have implications for the design of domain-specific sACE modulators.
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eLife Assessment
This important study shows, for the first time, the structure and snapshots of the dynamics of the full-length soluble Angiotensin-I converting enzyme dimer. The combination of structural and computational approaches elucidates with convincing evidence the conformational dynamics of the complex and key regions mediating the conformational change. This work provides an example of how conformational heterogeneity can be used to gain insights into protein function.
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Reviewer #1 (Public review):
Summary:
The authors report four cryoEM structures (2.99 to 3.65 Å resolution) of the 180 kDa, full-length, glycosylated, soluble Angiotensin-I converting enzyme (sACE) dimer, with two homologous catalytic domains at the N- and C-terminal ends (ACE-N and ACE-C). ACE is a protease capable of effectively degrading Aβ. The four structures are C2 pseudo-symmetric homodimers and provide insight into sACE dimerization. These structures were obtained using discrete classification in cryoSPARC and show different combinations of open, intermediate, and closed states of the catalytic domains, resulting in varying degrees of solvent accessibility to the active sites.
To deepen the understanding of the gradient of heterogeneity (from closed to open states) observed with discrete classification, the authors performed …
Reviewer #1 (Public review):
Summary:
The authors report four cryoEM structures (2.99 to 3.65 Å resolution) of the 180 kDa, full-length, glycosylated, soluble Angiotensin-I converting enzyme (sACE) dimer, with two homologous catalytic domains at the N- and C-terminal ends (ACE-N and ACE-C). ACE is a protease capable of effectively degrading Aβ. The four structures are C2 pseudo-symmetric homodimers and provide insight into sACE dimerization. These structures were obtained using discrete classification in cryoSPARC and show different combinations of open, intermediate, and closed states of the catalytic domains, resulting in varying degrees of solvent accessibility to the active sites.
To deepen the understanding of the gradient of heterogeneity (from closed to open states) observed with discrete classification, the authors performed all-atom MD simulations and continuous conformational analysis of cryo-EM data using cryoSPARC 3DVA, cryoDRGN, and RECOVAR. cryoDRGN and cryoSPARC 3DVA revealed coordinated open-closed transitions across four catalytic domains, whereas RECOVAR revealed independent motion of two ACE-N domains, also observed with cryoSPARC-focused classification. The authors suggest that the discrepancy in the results of the different methods for continuous conformational analysis in cryo-EM could result from different approaches used for dimensionality reduction and trajectory generation in these methods.
Strengths:
This is an important study that shows, for the first time, the structure and the snapshots of the dynamics of the full-length sACE dimer. Moreover, the study highlights the importance of combining insights from different cryo-EM methods that address questions difficult or impossible to tackle experimentally while lacking ground truth for validation.
Weaknesses:
The open, closed, and intermediate states of ACE-N and ACE-C in the four cryo-EM structures from discrete classification were designated quantitatively (based on measured atomic distances on the models fitted into cryo-EM maps, Figure 2D). Unfortunately, atomic models were not fitted into cryo-EM maps obtained with cryoSPARC 3DVA, cryoDRGN, and RECOVAR, and the open/closed states in these cases were designated based on qualitative analysis. As the authors clearly pointed out, there are many other methods for continuous conformational heterogeneity analysis in cryo-EM. Among these methods, some allow analyzing particle images in terms of atomic models, like MDSPACE (Vuillemot et al., J. Mol. Biol. 2023, 435:167951), which result in one atomic model per particle image and can help in analyzing cooperativity of domain motions through measuring atomic distances or angular differences between different domains (Valimehr et al., Int. J. Mol. Sci. 2024, 25: 3371). This could be discussed in the article.
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Reviewer #2 (Public review):
Summary:
The manuscript presents a valuable contribution to the field of ACE structural biology and dynamics by providing the first complete full-length dimeric ACE structure in four distinct states. The study integrates cryo-EM and molecular dynamics simulations to offer important insights into ACE dynamics. The depth of analysis is commendable, and the combination of structural and computational approaches enhances our understanding of the protein's conformational landscape. However, the strength of evidence supporting the conclusions needs refinement, particularly in defining key terms, improving structural validation, and ensuring consistency in data analysis. Addressing these points through major revisions will significantly improve the clarity, rigor, and accessibility of the study to a broader …
Reviewer #2 (Public review):
Summary:
The manuscript presents a valuable contribution to the field of ACE structural biology and dynamics by providing the first complete full-length dimeric ACE structure in four distinct states. The study integrates cryo-EM and molecular dynamics simulations to offer important insights into ACE dynamics. The depth of analysis is commendable, and the combination of structural and computational approaches enhances our understanding of the protein's conformational landscape. However, the strength of evidence supporting the conclusions needs refinement, particularly in defining key terms, improving structural validation, and ensuring consistency in data analysis. Addressing these points through major revisions will significantly improve the clarity, rigor, and accessibility of the study to a broader audience, allowing it to make a stronger impact in the field.
Strengths:
The integration of cryo-EM and MD simulations provides valuable insights into ACE dynamics, showcasing the authors' commitment to exploring complex aspects of protein structure and function. This is a commendable effort, and the depth of analysis is appreciated.
Weaknesses:
Several aspects of the manuscript require further refinement to improve clarity and scientific rigor as detailed in my recommendations for the authors.
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Reviewer #3 (Public review):
Summary:
Mancl et al. report four Cryo-EM structures of glycosylated and soluble Angiotensin-I converting enzyme (sACE) dimer. This moves forward the structural understanding of ACE, as previous analysis yielded partially denatured or individual ACE domains. By performing a heterogeneity analysis, the authors identify three structural conformations (open, intermediate open, and closed) that define the openness of the catalytic chamber and structural features governing the dimerization interface. They show that the dimer interface of soluble ACE consists of an N-terminal glycan and protein-protein interaction region, as well as C-terminal protein-protein interactions. Further heterogeneity mining and all-atom molecular dynamic simulations show structural rearrangements that lead to the opening and closing of …
Reviewer #3 (Public review):
Summary:
Mancl et al. report four Cryo-EM structures of glycosylated and soluble Angiotensin-I converting enzyme (sACE) dimer. This moves forward the structural understanding of ACE, as previous analysis yielded partially denatured or individual ACE domains. By performing a heterogeneity analysis, the authors identify three structural conformations (open, intermediate open, and closed) that define the openness of the catalytic chamber and structural features governing the dimerization interface. They show that the dimer interface of soluble ACE consists of an N-terminal glycan and protein-protein interaction region, as well as C-terminal protein-protein interactions. Further heterogeneity mining and all-atom molecular dynamic simulations show structural rearrangements that lead to the opening and closing of the catalytic pocket, which could explain how ACE binds its substrate. These studies could contribute to future drug design targeting the active site or dimerization interface of ACE.
Strengths:
The authors make significant efforts to address ACE denaturation on cryo-EM grids, testing various buffers and grid preparation techniques. These strategies successfully reduce denaturation and greatly enhance the quality of the structural analysis. The integration of cryoDRGN, 3DVA, RECOVAR, and all-atom simulations for heterogeneity analysis proves to be a powerful approach, further strengthening the overall experimental methodology.
Weaknesses:
In general, the findings are supported by experimental data, but some experimental details and approaches could be improved. For example, CryoDRGN analysis is limited to the top 5 PCA components for ease of comparison with cryoSPARC 3DVA, but wouldn't an expansion to more components with CryoDRGN potentially identify further conformational states? The authors also say that they performed heterogeneity analysis on both datasets but only show data for one. The results for the first dataset should be shown and can be included in supplementary figures. In addition, the authors mention that they were not successful in performing cryoSPARC 3DFLex analysis, but they do not show their data or describe the conditions they used in the methods section. These data should be added and clearly described in the experimental section.
Some cryo-EM data processing details are missing. Please add local resolution maps, box sizes, and Euler angle distributions and reference the initial PDB model used for model building.
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