SARS-CoV-2 S protein:ACE2 interaction reveals novel allosteric targets

  1. Palur V Raghuvamsi
  2. Nikhil K Tulsian
  3. Firdaus Samsudin
  4. Xinlei Qian
  5. Kiren Purushotorman
  6. Gu Yue
  7. Mary M Kozma
  8. Wong Y Hwa
  9. Julien Lescar
  10. Peter J Bond
  11. Paul A MacAry
  12. Ganesh S Anand

  • Curated by eLife

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    Summary: This is a timely and interesting exploration of the interaction between the Spike protein of SARS-CoV-2, the virus responsible for the COVID-19 pandemic, and the ACE2 receptor using hydrogen deuterium exchange mass spectrometry and molecular dynamics simulations. The Spike protein consists of two sub-domains S1 and S2 with the S1 needing to be cleaved-off so the S2 can become the fusion protein responsible for getting the SARS-CoV-2 into the cell. Structures are available but they do not shed light on how the protease furin can access the cleavage site between S1 and S2 in order to begin the process of fusion. The results suggest that the Spike-ACE2 interaction induces extremely long-range allosteric effects on the Spike protein that could trigger proteolysis of the Spike protein. Specifically, when ACE2 binds to the Spike protein, a conformational change occurs near the S1/S2 cleavage site, exposing it and likely making it more susceptible to furin cleavage. The binding also dampens exchange in the stalk region of the Spike protein. The authors refer to these regions as "dynamic hotspots in the pre-fusion state". The results of this work have implications for the development of small molecule inhibitors.

    In general, the work is timely, and the results will be of interest to many in the field. The major conclusions of the work are generally supported by the results.

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Abstract

The spike (S) protein is the main handle for SARS-CoV-2 to enter host cells via surface angiotensin-converting enzyme 2 (ACE2) receptors. How ACE2 binding activates proteolysis of S protein is unknown. Here, using amide hydrogen–deuterium exchange mass spectrometry and molecular dynamics simulations, we have mapped the S:ACE2 interaction interface and uncovered long-range allosteric propagation of ACE2 binding to sites necessary for host-mediated proteolysis of S protein, critical for viral host entry. Unexpectedly, ACE2 binding enhances dynamics at a distal S1/S2 cleavage site and flanking protease docking site ~27 Å away while dampening dynamics of the stalk hinge (central helix and heptad repeat [HR]) regions ~130 Å away. This highlights that the stalk and proteolysis sites of the S protein are dynamic hotspots in the prefusion state. Our findings provide a dynamics map of the S:ACE2 interface in solution and also offer mechanistic insights into how ACE2 binding is allosterically coupled to distal proteolytic processing sites and viral–host membrane fusion. Thus, protease docking sites flanking the S1/S2 cleavage site represent alternate allosteric hotspot targets for potential therapeutic development.

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  1. Version published to 10.7554/elife.63646 on eLife
  2. eLife

    Reviewer #3:

    This manuscript investigated the interactions of SARS-CoV-2 S protein and its RBD domain with ACE2 protein of host cells using mainly the HDX-MS approach. The results revealed the dynamics information about the interactions and how ACE2 binding at the RBD domain primes enhanced proteolytic processing at the S1/S2 site of S protein, and are potentially useful for the relevant research, e.g., therapeutic development. This is a rather straightforward study, without further biological validation of the major conclusions. Detailed comparison and integration of the HDX-MS results with those from cryo-EM were not provided in the manuscript as well. Some details of the manuscript also need further clarification.

    Major comments:

    1. Fig. S1: The SDS-PAGE showed around 90 kDa for the molecular weight of RBDisolated, which should be around 25 kDa based on its sequence (318-547). Please check and clarify.

    2. It is confusing about the existing forms of the S protein and ACE2 and their binding stoichiometry, regarding the statements such as "we measured dynamics of a trimer of this near-full length S protein..." (Page 4, line 87), "we performed HDXMS experiments of monomeric ACE2..." (Page 10, line 220-222), "......were pre-incubated at 37{degree sign}C for 30 min in a molar ratio of 1:1 to achieve >90% binding......" (Page S2, line 65-66). Please confirm whether the expressed ACE2 is dimeric and S protein is trimeric or not, and their binding stoichiometry is 1:1 or 2:3. Please also provide the concentration and calculation details for ensuring the >90% binding. If only one ACE2 in the ACE dimer and one S protein in the S protein trimer are involved in the binding, how sensitive and accurate could the HDX-MS results reflect the binding, since no HDX difference would be observed for the other ACE2 and other 2 S proteins?

    3. Page 2, line 33-35: Other studies (e.g., Ref. 11) have shown that ACE2 binding can enhance S1/S2 cleavage by furin and S1/S2 cleavage site could be possible targets for small molecule inhibitor/antibody development. It would be helpful if further evidence could be provided to support that the stalk hinge regions could also be the targets for that.

  3. eLife

    Reviewer #2:

    This is a super interesting exploration of the dynamic allosteric changes in the SARS-CoV-2 S protein upon engagement with the angiotensin 2 converting enzyme 2 (ACE2) receptor (and vice versa). It also represents a tour de force for HDX-MS since the S protein is almost 1200 amino acids long and the ACE2 is also very large. The data are beautiful and the analysis is well-done. The S protein consists of two sub-domains S1 and S2 with the S1 needing to be cleaved-off so the S2 can become the fusion protein responsible for getting the SARS-CoV-2 into the cell. Structures are available but they do not shed light on how the protease furin can access the cleavage site between S1 and S2 in order to begin the process of fusion. In this paper, the Anand group shows that when ACE2 binds to the S protein, a conformational change occurs near the S1/S2 cleavage site exposing it and likely making it more susceptible to furin cleavage. It also dampens exchange in the stalk region. They call these regions "dynamic hotspots in the pre-fusion state".

    There are some things that need to be addressed:

    1. The manuscript appears to have been hastily written, it would benefit from a scientific editor making it more readable. For example, line 90 ff "Average deuterium exchange at these 91 reporter peptides was monitored for comparative deuterium exchange analysis of S protein, ACE2 receptor and S:ACE2 complex, along with a specific ACE2 complex with the isolated RBD." Presumably "reporter peptides" refers to the 321 peptides mentioned two sentences earlier...Why is the ACE2 complex with the isolated RBD qualified as "specific" while none of the others are? Then the article continues with more information about glycosylation…

    2. Figure S1B the concentrations should be reported in molar not ng/ml

    3. Line 90 and Figure S2: A bit more should be said about the glycosylation sites. If only non-glycosylated peptides are observed in the pepsin digestion, the coverage map (Fig. S2), shows expected lack of coverage for only a few sites (17, 122, 149, 165, 234, 282, 709, 1134) whereas many other sites are covered by peptides. Does this indicate that these sites are mostly not glycosylated?

    4. Fig. S3 legend seems to indicate that uptake of each peptide is plotted, whereas uptake per residue should be plotted because overlapping peptides make these data misleading. The peptides are shown in the other relative uptake graphs, but then there is more than one data point per peptide. Can the authors explain a bit more in the legend how they got the data in these figures?

    5. Fig. S4 seems to indicate that the cleavage site is already very dynamic. Can the authors explain this?

    6. Line 98-99 "... Mapping the relative deuterium exchange across all peptides onto this S protein model showed the greatest deuterium exchange at the stalk region" seems to contradict lines 105-106 "The deuterium exchange heat map showed the highest relative exchange in the S2 subunit (Fig. S3) and helical segments," Please clarify.

    7. Fig. 2 A and B look like the same molecular structure (nice that they are in the same orientation) but the domains are labeled differently. Yet a third domain listing is used in panel E. Comparing panels A and B, it's a little strange that some of the least dynamic spots in the Head/ECD are the highest exchanging, do the authors want to comment on this?

    8. I thank the authors for the details provided in the Methods section regarding the HDX-MS data. If it wouldn't slow things down too much, it would be great if the RFU data were calculated after back exchange correction. Even an imperfect correction (such as a global correction for the back exchange during analysis) would make the data more meaningful.

    9. Fig. 3C and 3D look remarkably different considering that they both are reflecting the RBD:ACE2 interaction. Did the authors attempt to find a convergent set of peptides to do this analysis? Perhaps if the binding site were labeled it would help make the differences look less important (overall the top part of the molecule is blue and the bottom more-or-less has some red and if that's all we are supposed to get out of this figure then it is ok).

    10. Fig. 4. The authors state that the significance cut-off for difference in deuterium exchange is 0.3 D but I don't see where they explain how they derived this value.

  4. eLife

    Reviewer #1:

    The authors have used hydrogen deuterium exchange mass spectrometry and molecular dynamics simulations to study the interaction between the sars-cov-2 spike protein and the ace2 protein. The results suggest that the protein-protein interaction induces extremely long-range allosteric effects on the spike protein, triggering the proteolysis of the spike protein. The results of this work have implications for the development of small molecule inhibitors.

    In general, the manuscript is written extremely well. The work is timely, and the results will be of interest to many. The major conclusions of the work are generally supported by the results. However, there are several key - generally minor - details, enumerated below, the authors should provide in order to strengthen the manuscript and validity of the results.

    1. The authors should provide more technical details of the molecular dynamics simulations in the supplementary materials. Could the authors provide more details on the equilibration protocol? Was there any analysis done or metric used to assess whether the system was properly equilibrated? How often were snapshots of the trajectory saved for analysis? How many Na+ and Cl- ions were added to achieve 0.15 M of salt concentration? Also, how many water molecules were added? These details are relevant to the non-casual readers.

    2. The authors should probably include the techniques used to study the systems in the abstract section of the manuscript.

    3. Also, the authors should probably also include the fact that they performed molecular dynamics simulations in the last paragraph of the introduction. This is not apparent until toward the end of the first paragraph of the results and discussion sections.

    4. Page 7; line 147: Figure 4 is introduced before Figure 3. The authors should switch the order or modify accordingly.

    5. Figure S1: Could the authors elaborate on Figure S1B in the figure legend? Is (i) measuring the binding of ace2 to the S protein? Is (ii) measuring the binding of RBD to the ace2 protein? The distinction between (i) and (ii) is not made in the figure legend.

    In summary, the work is interesting and timely, and the manuscript will be of interest to many in the field. The authors should address the aforementioned points.

  5. eLife

    Summary: This is a timely and interesting exploration of the interaction between the Spike protein of SARS-CoV-2, the virus responsible for the COVID-19 pandemic, and the ACE2 receptor using hydrogen deuterium exchange mass spectrometry and molecular dynamics simulations. The Spike protein consists of two sub-domains S1 and S2 with the S1 needing to be cleaved-off so the S2 can become the fusion protein responsible for getting the SARS-CoV-2 into the cell. Structures are available but they do not shed light on how the protease furin can access the cleavage site between S1 and S2 in order to begin the process of fusion. The results suggest that the Spike-ACE2 interaction induces extremely long-range allosteric effects on the Spike protein that could trigger proteolysis of the Spike protein. Specifically, when ACE2 binds to the Spike protein, a conformational change occurs near the S1/S2 cleavage site, exposing it and likely making it more susceptible to furin cleavage. The binding also dampens exchange in the stalk region of the Spike protein. The authors refer to these regions as "dynamic hotspots in the pre-fusion state". The results of this work have implications for the development of small molecule inhibitors.

    In general, the work is timely, and the results will be of interest to many in the field. The major conclusions of the work are generally supported by the results.

  6. ScreenIT

    SciScore for 10.1101/2020.10.13.337212: (What is this?)

    Please note, not all rigor criteria are appropriate for all manuscripts.

    Table 1: Rigor

    Institutional Review Board Statementnot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variablenot detected.

    Table 2: Resources

    Software and Algorithms
    SentencesResources
    Materials and Methods:
    Methods
    suggested: None

    Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

    Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.

    Results from TrialIdentifier: No clinical trial numbers were referenced.

    Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

    Results from JetFighter: Please consider improving the rainbow (“jet”) colormap(s) used on pages 27, 13, 14 and 30. At least one figure is not accessible to readers with colorblindness and/or is not true to the data, i.e. not perceptually uniform.

    Results from rtransparent:
    • Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
    • Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
    • No protocol registration statement was detected.

    About SciScore

    SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

  7. Version published to 10.1101/2020.10.13.337212 on bioRxiv