1. Biochemistry and Chemical Biology
  2. Structural Biology and Molecular Biophysics
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SARS-CoV-2 S protein:ACE2 interaction reveals novel allosteric targets

  1. Palur V Raghuvamsi
  2. Nikhil Kumar Tulsian
  3. Firdaus Samsudin
  4. Xinlei Qian
  5. Kiren Purushotorman
  6. Gu Yue
  7. Mary M Kozma
  8. Wong Yee Hwa
  9. Julien Lescar
  10. Peter J Bond  Is a corresponding author
  11. Paul Anthony MacAry  Is a corresponding author
  12. Ganesh Srinivasan Anand  Is a corresponding author
  1. National University of Singapore, Singapore
  2. A*STAR, Singapore
  3. National Technological University, Singapore
Research Article
  • Cited 12
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Cite this article as: eLife 2021;10:e63646 doi: 10.7554/eLife.63646

Abstract

The Spike (S) protein is the main handle for SARS-CoV-2 to enter host cells via surface 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) regions ~130 Å away. This highlights that the stalk and proteolysis sites of the S protein are dynamic hotspots in the pre-fusion 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. Our findings highlight protease docking sites flanking the S1/S2 cleavage site, fusion peptide and heptad repeat 1 (HR1) as alternate allosteric hotspot targets for potential therapeutic development.

Data availability

All data generated or analysed during this study are included in the manuscript and supporting files. Source data files have been provided for Figures 2, 3, 4 and 5.

Article and author information

Author details

  1. Palur V Raghuvamsi

    Biological Sciences, National University of Singapore, Singapore, Singapore
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-0897-6935
  2. Nikhil Kumar Tulsian

    Biological Sciences, National University of Singapore, Singapore, Singapore
    Competing interests
    The authors declare that no competing interests exist.
  3. Firdaus Samsudin

    Bioinformatics Institute, A*STAR, Singapore, Singapore
    Competing interests
    The authors declare that no competing interests exist.
  4. Xinlei Qian

    Life Sciences Institute, National University of Singapore, Singapore, Singapore
    Competing interests
    The authors declare that no competing interests exist.
  5. Kiren Purushotorman

    Microbiology and Immunology, National University of Singapore, Singapore, Singapore
    Competing interests
    The authors declare that no competing interests exist.
  6. Gu Yue

    Microbiology and Immunology, National University of Singapore, Singapore, Singapore
    Competing interests
    The authors declare that no competing interests exist.
  7. Mary M Kozma

    Life Sciences Institute, National University of Singapore, Singapore, Singapore
    Competing interests
    The authors declare that no competing interests exist.
  8. Wong Yee Hwa

    School of Biological Sciences, National Technological University, Singapore, Singapore
    Competing interests
    The authors declare that no competing interests exist.
  9. Julien Lescar

    School of Biological Sciences, National Technological University, Singapore, Singapore
    Competing interests
    The authors declare that no competing interests exist.
  10. Peter J Bond

    Microbiology and Immunology, National University of Singapore, Singapore, Singapore
    For correspondence
    peterjb@bii.a-star.edu.sg
    Competing interests
    The authors declare that no competing interests exist.
  11. Paul Anthony MacAry

    Microbiology and Immunology, National University of Singapore, Singapore, Singapore
    For correspondence
    micpam@nus.edu.sg
    Competing interests
    The authors declare that no competing interests exist.
  12. Ganesh Srinivasan Anand

    Biological Sciences, National University of Singapore, Singapore, Singapore
    For correspondence
    gsa5089@psu.edu
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-8995-3067

Funding

Ministry of Education - Singapore (Research Fellowship)

  • Ganesh Srinivasan Anand

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Reviewing Editor

  1. Donald Hamelberg, Georgia State University, United States

Publication history

  1. Received: October 1, 2020
  2. Accepted: February 5, 2021
  3. Accepted Manuscript published: February 8, 2021 (version 1)
  4. Version of Record published: March 4, 2021 (version 2)

Copyright

© 2021, Raghuvamsi et al.

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.

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  1. Further reading

Further reading

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    A large number of inhibitory receptors recruit SHP1 and/or SHP2, tandem-SH2-containing phosphatases through phosphotyrosine-based motifs immunoreceptor tyrosine-based inhibitory motif (ITIM) and immunoreceptor tyrosine-based switch motif (ITSM). Despite the similarity, these receptors exhibit differential effector binding specificities, as exemplified by the immune checkpoint receptors PD-1 and BTLA, which preferentially recruit SHP2 and SHP1, respectively. The molecular basis by which structurally similar receptors discriminate SHP1 and SHP2 is unclear. Here, we provide evidence that human PD-1 and BTLA optimally bind to SHP1 and SHP2 via a bivalent, parallel mode that involves both SH2 domains of SHP1 or SHP2. PD-1 mainly uses its ITSM to prefer SHP2 over SHP1 via their C-terminal SH2 domains (cSH2): swapping SHP1-cSH2 with SHP2-cSH2 enabled PD-1:SHP1 association in T cells. In contrast, BTLA primarily utilizes its ITIM to prefer SHP1 over SHP2 via their N-terminal SH2 domains (nSH2). The ITIM of PD-1, however, appeared to be de-emphasized due to a glycine at pY+1 position. Substitution of this glycine with alanine, a residue conserved in BTLA and several SHP1-recruiting receptors, was sufficient to induce PD-1:SHP1 interaction in T cells. Finally, structural simulation and mutagenesis screening showed that SHP1 recruitment activity exhibits a bell-shaped dependence on the molecular volume of the pY+1 residue of ITIM. Collectively, we provide a molecular interpretation of the SHP1/SHP2-binding specificities of PD-1 and BTLA, with implications for the mechanisms of a large family of therapeutically relevant receptors.

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    Research Article Updated

    SNARE proteins have been described as the effectors of fusion events in the secretory pathway more than two decades ago. The strong interactions between SNARE domains are clearly important in membrane fusion, but it is unclear whether they are involved in any other cellular processes. Here, we analyzed two classical SNARE proteins, syntaxin 1A and SNAP25. Although they are supposed to be engaged in tight complexes, we surprisingly find them largely segregated in the plasma membrane. Syntaxin 1A only occupies a small fraction of the plasma membrane area. Yet, we find it is able to redistribute the far more abundant SNAP25 on the mesoscale by gathering crowds of SNAP25 molecules onto syntaxin clusters in a SNARE-domain-dependent manner. Our data suggest that SNARE domain interactions are not only involved in driving membrane fusion on the nanoscale, but also play an important role in controlling the general organization of proteins on the mesoscale. Further, we propose these mechanisms preserve active syntaxin 1A–SNAP25 complexes at the plasma membrane.