Structure of SARS-CoV-2 M protein in lipid nanodiscs
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Evaluation Summary:
This paper reports the structure of the M protein of SARS-CoV-2, as determined by cryoEM. The structure is well-determined and reveals a homodimer with overall similar structure as ORF3a, another virally encoded protein. The surface charge distribution is skewed towards positive at the C-terminal domain, which suggests roles in interactions with viral N and S proteins, and possibly viral RNA. The work is of relevance to virologists, especially those studying SARS-CoV-2.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)
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Abstract
SARS-CoV-2 encodes four structural proteins incorporated into virions, spike (S), envelope (E), nucleocapsid (N), and membrane (M). M plays an essential role in viral assembly by organizing other structural proteins through physical interactions and directing them to sites of viral budding. As the most abundant protein in the viral envelope and a target of patient antibodies, M is a compelling target for vaccines and therapeutics. Still, the structure of M and molecular basis for its role in virion formation are unknown. Here, we present the cryo-EM structure of SARS-CoV-2 M in lipid nanodiscs to 3.5 Å resolution. M forms a 50 kDa homodimer that is structurally related to the SARS-CoV-2 ORF3a viroporin, suggesting a shared ancestral origin. Structural comparisons reveal how intersubunit gaps create a small, enclosed pocket in M and large open cavity in ORF3a, consistent with a structural role and ion channel activity, respectively. M displays a strikingly electropositive cytosolic surface that may be important for interactions with N, S, and viral RNA. Molecular dynamics simulations show a high degree of structural rigidity in a simple lipid bilayer and support a role for M homodimers in scaffolding viral assembly. Together, these results provide insight into roles for M in coronavirus assembly and structure.
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Evaluation Summary:
This paper reports the structure of the M protein of SARS-CoV-2, as determined by cryoEM. The structure is well-determined and reveals a homodimer with overall similar structure as ORF3a, another virally encoded protein. The surface charge distribution is skewed towards positive at the C-terminal domain, which suggests roles in interactions with viral N and S proteins, and possibly viral RNA. The work is of relevance to virologists, especially those studying SARS-CoV-2.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)
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Reviewer #1 (Public Review):
The manuscript by Dolan et al. presents a high-resolution structure of the SARS-CoV-2 M (membrane) protein, determined by cryo-electron microscopy. Despite the protein's small size (50 kDa as a homodimer), the structure is well-determined and of sufficient resolution to build a confident model for the vast majority of the protein chain (missing only a short disordered N-terminal tail). The protein forms a homodimer with each protomer possessing three transmembrane helices and a beta-strand rich C-terminal domain. The overall structure of M is similar to that of ORF3a, a viral-encoded pore protein. The cytosolic surface of M's C-terminal domain is highly positively charged, and the authors propose that this charge mediates interactions with the N protein, S protein, and possibly viral RNA. Finally, the …
Reviewer #1 (Public Review):
The manuscript by Dolan et al. presents a high-resolution structure of the SARS-CoV-2 M (membrane) protein, determined by cryo-electron microscopy. Despite the protein's small size (50 kDa as a homodimer), the structure is well-determined and of sufficient resolution to build a confident model for the vast majority of the protein chain (missing only a short disordered N-terminal tail). The protein forms a homodimer with each protomer possessing three transmembrane helices and a beta-strand rich C-terminal domain. The overall structure of M is similar to that of ORF3a, a viral-encoded pore protein. The cytosolic surface of M's C-terminal domain is highly positively charged, and the authors propose that this charge mediates interactions with the N protein, S protein, and possibly viral RNA. Finally, the authors perform molecular dynamics simulations that demonstrate that the M dimer is relatively stable, at least over the time-frame of the simulation (1.6 microseconds).
Overall, this is a straightforward work that describes the structure of an important protein in the life cycle of SARS-CoV-2. As such, it is important and timely, and will be of interest to a broad set of readers. The work suggests many directions for future experiments.
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Reviewer #2 (Public Review):
The SARS-CoV-2 M protein is an abundant structural protein in the viral envelope and is a potential target for vaccine and therapeutic development. In this study, Dolan and colleagues present the single-particle cryo-electron microscopy structure of the M protein in lipid nanodiscs. M forms a dimer structure, similar to the previously characterized SARS-CoV-2 ORF3a viroporin, which was proposed to function as an ion channel. Structural analysis of M and molecular dynamics simulations provide a plausible explanation of why M functions as a structural scaffold protein rather than an ion channel. The conclusions of this paper are generally supported by structural analysis and molecular dynamics simulations.
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This Zenodo record is a permanently preserved version of a PREreview. You can view the complete PREreview at https://prereview.org/reviews/7086808.
SUMMARY –
COVID-19 disease is a global pandemic caused by the SARS-CoV-2 virus. Viral protein structures such as Spike, and RNA-dependant RNA polymerase has played a key role in developing structure-guided antibodies and drug designing. Kimberly Dolan and colleagues have made a significant step by unravelling the structure of Membrane (M) protein in a lipid environment by Cryo-EM. M protein plays an essential role in viral assembly and the protein structure shed light on the architecture and features important for protein function. The structure of M protein is very similar to another SARS-CoV-2 protein ORF3a with some key differences that the authors have discussed extensively.
STRE…
This Zenodo record is a permanently preserved version of a PREreview. You can view the complete PREreview at https://prereview.org/reviews/7086808.
SUMMARY –
COVID-19 disease is a global pandemic caused by the SARS-CoV-2 virus. Viral protein structures such as Spike, and RNA-dependant RNA polymerase has played a key role in developing structure-guided antibodies and drug designing. Kimberly Dolan and colleagues have made a significant step by unravelling the structure of Membrane (M) protein in a lipid environment by Cryo-EM. M protein plays an essential role in viral assembly and the protein structure shed light on the architecture and features important for protein function. The structure of M protein is very similar to another SARS-CoV-2 protein ORF3a with some key differences that the authors have discussed extensively.
STRENGTHS –
1) Single particle Cryo-EM of small proteins less than 100KDa is a challenging task but the authors were successfully able to stabilize the 50KDa dimeric M protein in lipid nanodisc for structural study.
2) The paper discusses in-depth the structural comparison between the M and ORF3a proteins that are structurally similar but perform different roles in the SARS-Cov-2 virus. While M protein is essential for viral assembly, ORF3a acts as a non-selective calcium channel.
MAJOR CONCERNS –
1) The M protein helps in viral assembly but the paper does not have any in-vitro interaction study with other viral proteins. Interaction study of the M protein with other structural proteins of SARS-CoV-2 would have helped understand the molecular basis of interaction between the structural proteins. 2) The authors did a comparative structural analysis of the M and ORF-3a but a calcium uptake assay with ORF3a as a positive control could have led to additional experimental data to verify the difference in the function of two structurally homologous proteins.
3) A recent study by Zhikuan Zhang et al., showed that the M protein overexpressed in mammalian cells purified in GDN showed higher order oligomers ranging from dimeric to hexameric organization. I think that the detergent or the expression system could have affected the oligomerization behavior of M protein in two different conditions. It would be interesting to see if the M protein overexpressed in sf9 cells and purified in GDN shows similar characteristics.
MINOR CONCERNS –
1) The experimental protein structure is compared with the AlphaFold structure but there is no calculated RMSD value assigned to highlight the significant difference between the two structures.
2) In the Method section the FSEC was done on the superose 6 column but the column dimensions are not mentioned. Also in the FSEC Figure S1-(a) the X-axis represents the retention time, which can be modified to elution volume to match the Figures S1-(b),(c) for easy comparison between the FSEC profile with the large-scale size-exclusion profile of M protein in detergent and nanodisc condition.
3) The data processing can be represented as a Cryo-EM processing pipeline to easily track the data processing from the initial micrographs to complete 3D structure and validation.
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