Timeline of changes in spike conformational dynamics in emergent SARS-CoV-2 variants reveal progressive stabilization of trimer stalk with altered NTD dynamics
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Curated by eLife
eLife assessment
This fundamental and timely study provides insights into the structural dynamics of several relevant mutant forms of SARS-CoV-2 spike protein, including the most recent omicron variant. The hydrogen/deuterium-exchange studies provide compelling evidence for the stabilization of the spike stalk in conjunction with increased dynamics of the N-terminal domain, where binding to the ACE2 receptor occurs. These results have profound implications for the development of small molecule inhibitors of the spike protein-ACE2 receptor interaction.
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Abstract
SARS-CoV-2 emergent variants are characterized by increased viral fitness and each shows multiple mutations predominantly localized to the spike (S) protein. Here, amide hydrogen/deuterium exchange mass spectrometry has been applied to track changes in S dynamics from multiple SARS-CoV-2 variants. Our results highlight large differences across variants at two loci with impacts on S dynamics and stability. A significant enhancement in stabilization first occurred with the emergence of D614G S followed by smaller, progressive stabilization in subsequent variants. Stabilization preceded altered dynamics in the N-terminal domain, wherein Omicron BA.1 S showed the largest magnitude increases relative to other preceding variants. Changes in stabilization and dynamics resulting from S mutations detail the evolutionary trajectory of S in emerging variants. These carry major implications for SARS-CoV-2 viral fitness and offer new insights into variant-specific therapeutic development.
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Author Response
Reviewer #1 (Public Review):
In this manuscript, Braet et al provide a rigorous analysis of SARS-CoV-2 spike protein dynamics using hydrogen/deuterium exchange mass spectrometry. Their findings reveal an interesting increase in the dynamics of the N-terminal domain that progressed with the emergence of new variants. In addition, the authors also observe an increase in the stabilization of the spike trimeric core, which they identify originates from the early D614G mutation.
Overall this is a timely and interesting exploration of spike protein dynamics, which have so far remained largely unexplored in the literature.
What I find a bit missing in this manuscript is a link between how the identified changes in protein dynamics lead to increased viral fitness. While there are some possibilities listed in the …
Author Response
Reviewer #1 (Public Review):
In this manuscript, Braet et al provide a rigorous analysis of SARS-CoV-2 spike protein dynamics using hydrogen/deuterium exchange mass spectrometry. Their findings reveal an interesting increase in the dynamics of the N-terminal domain that progressed with the emergence of new variants. In addition, the authors also observe an increase in the stabilization of the spike trimeric core, which they identify originates from the early D614G mutation.
Overall this is a timely and interesting exploration of spike protein dynamics, which have so far remained largely unexplored in the literature.
What I find a bit missing in this manuscript is a link between how the identified changes in protein dynamics lead to increased viral fitness. While there are some possibilities listed in the discussion, I think these should be elaborated upon further. In addition, it should also be discussed how understanding the changes in the spike protein dynamics could have implications for the development of small molecule inhibitors for the virus.
We have included information in the introduction and conclusion to make the connection more clearly between our observations, function, and viral fitness of spike protein. We have also connected specific mutations to observed function. We have re-organized the discussion for increased clarity and to improve the correlation of our observations to viral fitness.
Reviewer #2 (Public Review):
The study systematically looks at dynamic differences across variants longitudinally and the authors appropriately only limit their analyses to peptides that are conserved across the different variants.
There are some concerns listed below, particularly related to the ensemble heterogeneity that is reported and need considerable revision.
- The authors explain that cold-temperature treatment of the S trimer ectodomain constructs has been shown to lead to instability and heterogeneity. They also show this with a comparison of untreated vs. 3-hour 37 ℃ treated samples. I'm confused as to why "During automated HDXMS experiments protein samples were stored at 0 ℃". Will this not cause issues in protein heterogeneity, where the longer the protein sits at 0 ℃ the more potential heterogeneity there will be, and thus greatly confound the analysis?
We thank the reviewer for highlighting this point. We have carefully examined and reevaluated our analysis of both wild -type and variant spike HDXMS. During automated HDXMS experiments, protein samples are indeed maintained at 0 ℃, in between runs and replicates for fixed periods of time (4 h per replicate). In the case of WT S, we did observe conformational heterogeneity between replicates (Figure 2- figure supplement 6), as correctly pointed out by the reviewer. We have repeated analysis of WT S without 0 ℃ incubation in automated HDXMS experiments. In the revised manuscript, Figure 2 shows the more homogenous conformation of WT S, when not incubated at 0 ℃ in between replicates. Extension of these analyses to D614G (Figure 2- figure supplement 7) and all subsequent variants that each contain D614G, showed almost no conformational heterogeneity.
We have included a detailed description (lines 237-244) of the revised manuscript to describe in greater detail effects of 0 ℃ incubation on HDXMS of WT S.
Our results revealed that WT S was more sensitive to cold denaturation as described previously [Costello et al. 2021] where the reported half-life for conformational transitions after 0 ℃ incubation was 17 hours. We had not anticipated conformational heterogeneity revealed by deuterium exchange when using an automated HDXMS setup. Upon further review, we see a significant ensemble shift in trimer stalk peptides for the second and third replicates which sat at 0 ℃ for 4 and 8 hours respectively. This is only observed in WT but not any of the other variant S samples. We thank the reviewer for pointing this out and strengthening our conclusions.
- The authors presume that the bimodal spectra that are observed reflect EX1 kinetics, however, there can be multiple reasons for an apparent bimodal distribution in the spectra. I agree that some of the spectra indicate that more than a single species is present, but what the two populations represent is murky. In Figure 2D, the apparent size of the highly deuterated population gets larger going from the 60 sec to the 600-sec spectra, as expected for an EX1 transition. However, in Figure 3D the WT highly deuterated population gets smaller going from the 60-sec to the 600-sec spectra. Were bimodal examples observed beyond those shown in Figure 2?
We agree with the reviewer. The appearance of bimodal spectra in deuterium exchange of S protein peptides in WT S are not a result of EX1 kinetics alone. We have revised the explanation for the presence of the bimodal spectra. These are largely a consequence of automated HDXMS workflows, that included 0° C incubations for short periods of time in between replicates. We report new experiments where we have eliminated 0 °C incubations by incubating at 20 °C between replicates and observed a lot lower conformational heterogeneity.
Consequently, the shifts in bimodal spectra in figure 3D for WT S are also likely a consequence of automated HDX MS experiments with 0 ℃ incubation. We have carried out new experiments without 0 ℃ incubation, and these are shown in a revised figure 3. Even without 0 ℃ incubation, we do see bimodal spectra for certain peptides [figure 2 – S5]. These reflect an ensemble of prefusion and splayed conformations of WT S. Lack of baseline resolution precludes application of HDexaminer to resolve spectral envelopes quantitatively.
- How were the spectra that appeared broadened analyzed? There is no description of this in the methods, and the only data shown for this is in table 1. The left/right percentages are reported without any description of how they were obtained. Are these solely from a single spectrum? The most alarming issue is that Table 1B reports 9.4% for the right population of the 988-998 peptide, but the corresponding spectra in Figure 3D doesn't seem to have any highly deuterated population at all.
We agree with the reviewer. We have removed HD examiner analysis of spectral broadening. Some of the spectral broadening was a consequence of 0 ℃ incubation in automated HDX analyses. These have been revised in new supplemental figures for wild -type HDX MS. Baseline resolution precludes effective quantitation of spectral envelopes, Figure 2-figure supplement 5 highlights qualitatively the spectral broadening for the reader’s benefit.
- The authors state on page 12: "Replicate analysis of stabilized S trimers with incubation at 4C prior to deuterium exchange (see methods) showed a time-dependent reversal of stabilization as reported previously (Costello et al., 2022), most evident at the same peptides." Is this data shown anywhere? If not then it should be included somewhere, possibly in table 1 as I would expect the cold treatment to offset the left/right population sizes.
We note that this statement was misleading and have revised the text. The time-dependent reversal of stabilization has previously been described (Costello et al., 2022 paper) and is not part of this study.
- The authors state that peptide 899-913 'exhibits a slow conformational interconversion (time scale ~ 15-30 min)'. Where did this estimated rate come from? From the data shown and the limited number of time points, I don't think there is sufficient sampling of this conformational transition to really narrow down the exact timescale, especially since the ratio of left/right populations is so dependent on the pre-treatment of the sample prior to deuterium exchange. (See 1st comment)
We thank the reviewer. The heterogeneity in deuterium exchange is attributable to the variable 0 °C incubation times in our automated HDXMS workflow. We have removed any explanations of conformational interconversion occurring in our experimental timescales.
- The woods plots presented in the Supporting information: (Figures 2-S4, 2-S5, 3-S4, 4-S2, 5-S2, 6-S2) are not conventional Woods plots. Normally the plots would indicate a global threshold for what is deemed to be significant based on the overall error in the dataset. From what I gather the authors used error within an individual peptide to establish significance for each specific peptide, which would be okay, but the authors don't describe the number of replicates or how the p-value was calculated. I would strongly recommend that the authors instead rely on a hybrid significance testing approach, as described recently: (PMID 31099554). What's really alarming with the current approach is that several of the Woods plots shown have data points found to be significantly different that are right at zero on the y-axis.
We thank the reviewer. We have replaced all of the Woods plots with volcano plots. We have now applied a hybrid significance testing approach as recommended by the reviewer.
- Table 1: The summary of the peptides with observed bimodal behavior should include data from the replicates, particularly for assessment of how consistent the left/right population sizes are across replicates. Instead of just a percentage, the table should report an average and the standard deviation from the replicate measurements. Furthermore, the table should also include peptides that are overlapping with those presented. Based on Figure 2-figure supplement 1, there are at least two other peptides that cover the 899-913 region. These additional peptides should show a similar trend with bimodal profiles and will be important for showing how reproducible the apparent EX1 kinetics are in the dataset.
All available replicates and overlapping peptides should be analyzed to ensure that these percentages reported are consistent across the data. It is also odd that the authors choose to use the 3+ charge state of the WT, but the 2+ for the D614G mutant. If both charge states were present, then both of them should be analyzed to ensure the population distributions are consistent within different charge states.
We thank the reviewers for their suggestion. We have removed Table 1 since bimodal spectra are not resolvable for quantitation as described previously. We instead show spectra of overlapping peptides in these regions for interpretation by the reader.
We show charge states that provide highest intensity for the peptides (Figure 2-figure supplement 5, Figure 3-figure supplement 3, Figure 4-figure supplement 3, Figure 5-figure supplement 3, Figure 6-figure supplement 3).
- The method for calculating p-values used to assess the significance of a difference in observed deuterium uptake is not described. The manuscript mentions technical replicates, but no specific information as to how many replicates were collected for each time point. These details should be included as they are also part of the summary table that is recommended for the publication of HDX data.
We have utilized hybrid significance testing as suggested by the reviewers to determine significance as outlined by Hageman et al. We have included this in table S3 and in the text.
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eLife assessment
This fundamental and timely study provides insights into the structural dynamics of several relevant mutant forms of SARS-CoV-2 spike protein, including the most recent omicron variant. The hydrogen/deuterium-exchange studies provide compelling evidence for the stabilization of the spike stalk in conjunction with increased dynamics of the N-terminal domain, where binding to the ACE2 receptor occurs. These results have profound implications for the development of small molecule inhibitors of the spike protein-ACE2 receptor interaction.
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Reviewer #1 (Public Review):
In this manuscript, Braet et al provide a rigorous analysis of SARS-CoV-2 spike protein dynamics using hydrogen/deuterium exchange mass spectrometry. Their findings reveal an interesting increase in the dynamics of the N-terminal domain that progressed with the emergence of new variants. In addition, the authors also observe an increase in the stabilization of the spike trimeric core, which they identify originates from the early D614G mutation.
Overall this is a timely and interesting exploration of spike protein dynamics, which have so far remained largely unexplored in the literature.
What I find a bit missing in this manuscript is a link between how the identified changes in protein dynamics lead to increased viral fitness. While there are some possibilities listed in the discussion, I think these should …Reviewer #1 (Public Review):
In this manuscript, Braet et al provide a rigorous analysis of SARS-CoV-2 spike protein dynamics using hydrogen/deuterium exchange mass spectrometry. Their findings reveal an interesting increase in the dynamics of the N-terminal domain that progressed with the emergence of new variants. In addition, the authors also observe an increase in the stabilization of the spike trimeric core, which they identify originates from the early D614G mutation.
Overall this is a timely and interesting exploration of spike protein dynamics, which have so far remained largely unexplored in the literature.
What I find a bit missing in this manuscript is a link between how the identified changes in protein dynamics lead to increased viral fitness. While there are some possibilities listed in the discussion, I think these should be elaborated upon further. In addition, it should also be discussed how understanding the changes in the spike protein dynamics could have implications for the development of small molecule inhibitors for the virus. -
Reviewer #2 (Public Review):
The study systematically looks at dynamic differences across variants longitudinally and the authors appropriately only limit their analyses to peptides that are conserved across the different variants.
There are some concerns listed below, particularly related to the ensemble heterogeneity that is reported and need considerable revision.
The authors explain that cold-temperature treatment of the S trimer ectodomain constructs has been shown to lead to instability and heterogeneity. They also show this with a comparison of untreated vs. 3-hour 37 C treated samples. I'm confused as to why "During automated HDXMS experiments protein samples were stored at 0 degrees". Will this not cause issues in protein heterogeneity, where the longer the protein sits at 0 C the more potential heterogeneity there will be, and …
Reviewer #2 (Public Review):
The study systematically looks at dynamic differences across variants longitudinally and the authors appropriately only limit their analyses to peptides that are conserved across the different variants.
There are some concerns listed below, particularly related to the ensemble heterogeneity that is reported and need considerable revision.
The authors explain that cold-temperature treatment of the S trimer ectodomain constructs has been shown to lead to instability and heterogeneity. They also show this with a comparison of untreated vs. 3-hour 37 C treated samples. I'm confused as to why "During automated HDXMS experiments protein samples were stored at 0 degrees". Will this not cause issues in protein heterogeneity, where the longer the protein sits at 0 C the more potential heterogeneity there will be, and thus greatly confound the analysis?
The authors presume that the bimodal spectra that are observed reflect EX1 kinetics, however, there can be multiple reasons for an apparent bimodal distribution in the spectra. I agree that some of the spectra indicate that more than a single species is present, but what the two populations represent is murky. In Figure 2D, the apparent size of the highly deuterated population gets larger going from the 60 sec to the 600-sec spectra, as expected for an EX1 transition. However, in Figure 3D the WT highly deuterated population gets smaller going from the 60-sec to the 600-sec spectra. Were bimodal examples observed beyond those shown in Figure 2?
How were the spectra that appeared broadened analyzed? There is no description of this in the methods, and the only data shown for this is in table 1. The left/right percentages are reported without any description of how they were obtained. Are these solely from a single spectrum? The most alarming issue is that Table 1B reports 9.4% for the right population of the 988-998 peptide, but the corresponding spectra in Figure 3D doesn't seem to have any highly deuterated population at all.
The authors state on page 12: "Replicate analysis of stabilized S trimers with incubation at 4C prior to deuterium exchange (see methods) showed a time-dependent reversal of stabilization as reported previously (Costello et al., 2022), most evident at the same peptides." Is this data shown anywhere? If not then it should be included somewhere, possibly in table 1 as I would expect the cold treatment to offset the left/right population sizes.
The authors state that peptide 899-913 'exhibits a slow conformational interconversion (time scale ~ 15-30 min)'. Where did this estimated rate come from? From the data shown and the limited number of time points, I don't think there is sufficient sampling of this conformational transition to really narrow down the exact timescale, especially since the ratio of left/right populations is so dependent on the pre-treatment of the sample prior to deuterium exchange. (See 1st comment)
The woods plots presented in the Supporting information: (Figures 2-S4, 2-S5, 3-S4, 4-S2, 5-S2, 6-S2) are not conventional Woods plots. Normally the plots would indicate a global threshold for what is deemed to be significant based on the overall error in the dataset. From what I gather the authors used error within an individual peptide to establish significance for each specific peptide, which would be okay, but the authors don't describe the number of replicates or how the p-value was calculated. I would strongly recommend that the authors instead rely on a hybrid significance testing approach, as described recently: (PMID 31099554). What's really alarming with the current approach is that several of the Woods plots shown have data points found to be significantly different that are right at zero on the y-axis.
Table 1: The summary of the peptides with observed bimodal behavior should include data from the replicates, particularly for assessment of how consistent the left/right population sizes are across replicates. Instead of just a percentage, the table should report an average and the standard deviation from the replicate measurements. Furthermore, the table should also include peptides that are overlapping with those presented. Based on Figure 2-figure supplement 1, there are at least two other peptides that cover the 899-913 region. These additional peptides should show a similar trend with bimodal profiles and will be important for showing how reproducible the apparent EX1 kinetics are in the dataset.
All available replicates and overlapping peptides should be analyzed to ensure that these percentages reported are consistent across the data. It is also odd that the authors choose to use the 3+ charge state of the WT, but the 2+ for the D614G mutant. If both charge states were present, then both of them should be analyzed to ensure the population distributions are consistent within different charge states.The method for calculating p-values used to assess the significance of a difference in observed deuterium uptake is not described. The manuscript mentions technical replicates, but no specific information as to how many replicates were collected for each time point. These details should be included as they are also part of the summary table that is recommended for the publication of HDX data.
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Reviewer #3 (Public Review):
The authors use hydrogen-deuterium exchange mass spectrometry (HDXMS) to assess the dynamics of several relevant mutant forms of SARS-CoV 2 Spike protein including the most recent Omicron variant. The Spike protein is heavily glycosylated and is a trimer so is a very difficult protein to study by HDXMS. The authors confirm the glycosylation sites, which can't be covered by the HDXMS experiment, yet they still manage to cover nearly 50% of the sequence revealing many interesting changes in dynamics in the prevalent circulating mutant forms. The beautiful HDXMS data reveal consistent trends as SARS-CoV2 mutates to survive including stabilization of the stalk and increased dynamics of the N-terminal domain where ACE2 receptor binding occurs. The authors incubate the protein at 37C and discover additional …
Reviewer #3 (Public Review):
The authors use hydrogen-deuterium exchange mass spectrometry (HDXMS) to assess the dynamics of several relevant mutant forms of SARS-CoV 2 Spike protein including the most recent Omicron variant. The Spike protein is heavily glycosylated and is a trimer so is a very difficult protein to study by HDXMS. The authors confirm the glycosylation sites, which can't be covered by the HDXMS experiment, yet they still manage to cover nearly 50% of the sequence revealing many interesting changes in dynamics in the prevalent circulating mutant forms. The beautiful HDXMS data reveal consistent trends as SARS-CoV2 mutates to survive including stabilization of the stalk and increased dynamics of the N-terminal domain where ACE2 receptor binding occurs. The authors incubate the protein at 37C and discover additional stabilization of the trimer occurs under these conditions explaining a lot of conflicting data in the literature done at different temperatures. These results have profound implications for the development of small molecule inhibitors of the Spike protein-ACE2 interaction.
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