Coronaviruses have long infected humans, yet none caused the same devastation as SARS-CoV-2 has (Li et al., 2020; Huang et al., 2020). For instance, a virulent and lethal coronavirus, SARS-CoV-1, caused a much smaller outbreak in 2002–2003 (Lee et al., 2003; Peiris et al., 2003). Numerous human coronaviruses such as NL63-CoV cause common colds annually (Fouchier et al., 2004; van der Hoek et al., 2004). With an intermediate virulence, SARS-CoV-2 causes a fatality rate that is significantly lower than that of SARS-CoV-1, but much higher than that of NL63-CoV. Because of its intermediate virulence, SARS-CoV-2 carriers show clinical signs that facilitate the spread of the virus: they may develop mild or no symptoms, experience delayed onset of symptoms, develop low levels of neutralizing antibodies, or endure prolonged virus shedding period (Wu et al., 2020; Zhou et al., 2020; Wölfel et al., 2020; Gao et al., 2020; Kronbichler et al., 2020). These features contribute to the wide spread of SARS-CoV-2 and severe health outcomes, triggering a global COVID-19 pandemic that was unprecedented in the era of modern medicine. Understanding the molecular determinants of COVID-19 is crucial for comprehending the recent pandemic and preventing future ones.

SARS-CoV-2 spike protein played a central role in the COVID-19 pandemic. It guides viral entry into host cells and is also a major target for the host immune responses (Du et al., 2009; Li, 2016). On newly packaged virus particles, the trimeric spike protein has a pre-fusion structure in which three receptor-binding S1 subunits sit on top of a trimeric membrane-fusion S2 stalk (Figure 1A). During viral entry, a receptor-binding domain (RBD) in S1 binds to a receptor on host cell surface for viral attachment and S2 switches to a post-fusion structure for the fusion of viral and host membranes (Li, 2016; Li, 2015). SARS-CoV-2, SARS-CoV-1, and NL63-CoV can all use ACE2 as their receptor (Li et al., 2003; Li et al., 2005; Shang et al., 2020c; Wan et al., 2020a; Wu et al., 2009). Early in the pandemic, we described three distinct characteristics of SARS-CoV-2 spike: its exceptional binding affinity for human ACE2, its proteolytic activation by human protease furin, and its existence as a mixture of two conformations: an open conformation in which the RBD is exposed and accessible to ACE2 and a closed conformation in which the RBD is buried and inaccessible to ACE2 (Shang et al., 2020b). While much has been learned about the first two characteristics (Shang et al., 2020c; Lan et al., 2020; Essalmani et al., 2022; Peacock et al., 2021; Geng et al., 2022; Zhang et al., 2023), the third still remains poorly understood. Interestingly, SARS-CoV-1 spike is predominantly in the open conformation, whereas NL63-CoV spike remains closed (Walls et al., 2016; Wrobel et al., 2020; Gui et al., 2017). Thus, two key questions arise: what molecular switches regulate the conformation of coronavirus spikes and how do the conformational changes influence coronaviruses’ infectivity and host immune responses?

Figure 1

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In this study, we used cryo-EM and biochemical approaches to identify spike residue 417 as a molecular switch that regulates the conformation of SARS-CoV-2 spike. We delved deeper into how this molecular switch affects receptor binding, viral entry, and immune evasion by SARS-CoV-2. Through regulation of its spike’s conformations, SARS-CoV-2 may have struck a balance between infection potency and immune evasiveness.

To understand the molecular mechanism that controls the spike’s switching between open and closed conformations, we compared the sequences of SARS-CoV-2 and SARS-CoV-1 spikes in the context of their tertiary structures. We identified residue 417 as potentially a key difference between the two spikes: in the closed SARS-CoV-2 trimeric spike, Lys417 in the RBD from one spike subunit forms a hydrogen bond with the main chain of Asn370 in the RBD from another spike subunit, stabilizing the RBDs in the closed conformation (Walls et al., 2020; Figure 1B). In SARS-CoV-1 spike, however, this residue becomes a valine (Li et al., 2005; Shang et al., 2020a; Figure 1C), losing its capability to form the hydrogen bond with another spike subunit and potentially destabilizing the closed conformation of the spike. Thus, we hypothesized that a K417V mutation causes more SARS-CoV-2 spike molecules to take the open conformation. To test this hypothesis, we investigated the impact of the K417V mutation on the structure and function of SARS-CoV-2 spike using a combination of cryo-EM and biochemical approaches.

To examine how the K417V mutation affects the conformation of SARS-CoV-2 spike, we performed cryo-EM analysis of the RBD conformation of SARS-CoV-2 spike ectodomain containing either Lys417 or Val417. To this end, we introduced the K417V mutation into SARS-CoV-2 spike ectodomain and purified both versions of the protein: K417-spike containing Lys417 and V417-spike containing Val417. Due to the lack of the transmembrane anchor, the recombinant spike ectodomain was intrinsically unstable. To stabilize the recombinant spike ectodomain, we introduced six proline mutations into its S2 subunit to lock up its pre-fusion structure, introduced mutations to its furin motif to prevent it from being cleaved during molecular maturation, and added a C-terminal foldon tag to facilitate its trimerization (Shang et al., 2020b; Hsieh et al., 2020; Ye et al., 2022). We successfully obtained the stabilized K417-spike and V417-spike and collected cryo-EM data on both proteins (Figure 2—figure supplements 1 and 2; Supplementary file 1). For the K417-spike, 55% of the molecules were in the closed conformation with all three RBDs down, whereas the rest of the molecules (45%) were in the open conformation with one RBD up and two RBDs down (Figure 2A and B). In contrast, for the V417-spike, these numbers became 32 and 68% for the closed and open conformations, respectively (Figure 2C and D). Therefore, the K417V mutation allowed more SARS-CoV-2 spike molecules to take the open conformation, confirming our hypothesis.

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While the above cryo-EM approach examined the conformation of recombinant spike ectodomain, we further examined the impact of the K417V mutation on the membrane-anchored and full-length SARS-CoV-2 spike that do not contain S2 or furin motif mutations. It has been shown that ACE2 only binds to the standing-up RBD in the open spike (Gui et al., 2017; Ye et al., 2021), suggesting that the open spike conformation is necessary for ACE2 binding. Hence, we characterized the capabilities of K417-spike and V417-spike in binding ACE2. First, we expressed the K417-spike and V417-spike on human cell surface and performed a protein pull-down assay using recombinant human ACE2 as the bait and the cell-surface-anchored spikes as the target (Figure 3A). For cross-validation, both His-tagged ACE2 and Fc-tagged ACE2 were used. We previously showed that this pull-down assay is a reliable method to probe the RBD conformation of cell-surface-anchored spikes, with higher pull-down levels of spikes associated with more spike molecules in the open conformation (Shang et al., 2020b). Western blot analysis showed that some of the spike molecules had been cleaved at the S1/S2 boundary, confirming the intact furin motif (Figure 3A). The pull-down result showed that, compared to K417-spike, more V417-spike molecules had been pulled down by ACE2, suggesting that V417-spike had an increased probability of binding to ACE2 (Figure 3A). Second, we conducted a flow cytometry assay to detect the interactions between recombinant human ACE2 and cell-surface-anchored spikes (Figure 3B, Figure 3—figure supplement 1). Here, two versions of both K417-spike and V417-spike were used: one containing the intact furin motif and the other the inactivated furin motif. The result showed that V417-spike binds to ACE2 better than does K417-spike, no matter whether the furin motif was intact or inactivated (Figure 3B, Figure 3—figure supplement 1). Taken together, both protein pull-down and flow cytometry results reveal that the K417V mutation allowed more SARS-CoV-2 spike molecules to take the open conformation, confirming the cryo-EM data and our hypothesis.

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Numerical data for Figure 3B.

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Numerical data for Figure 3C.

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Raw image for Figure 3C.

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To understand the role of the spike’s conformations in viral entry, we conducted a pseudovirus entry assay. Specifically, retroviruses pseudotyped with SARS-CoV-2 spike (i.e., SARS-CoV-2 pseudoviruses) were evaluated for their capability to enter ACE2-expressing human cells. Two types of SARS-CoV-2 pseudoviruses were used: the K417-spike-charged pseudoviruses (i.e., K417-pseudoviruses) and V417-spike-charged pseudoviruses (V417-pseudoviruses). Both types of pseudoviruses contain the furin motif in their spike. Western blot analysis showed that some of the spike molecules had been cleaved at the S1/S2 boundary when expressed on pseudovirus surface, confirming the intact furin motif (Figure 3C). The pseudovirus entry result showed that V417-pseudoviruses entered cells more efficiently than did K417-pseudoviruses (Figure 3C). Thus, the open spike enhances not only ACE2 binding, but also viral entry.

To understand the role of the spike’s conformation in immune evasion, we analyzed how neutralizing antibodies recognize the RBD in the context of the trimeric spike protein. To this end, we summarized the binding sites of known neutralizing antibodies on the RBD (Figure 4A). These neutralizing antibodies were discovered individually from COVID-19 patients. They generally recognize five groups of neutralizing epitopes on the RBD (Starr et al., 2021; Dussupt et al., 2021; Scheid et al., 2021; Fedry et al., 2021). Three groups of these RBD epitopes are only accessible to neutralizing antibodies when the spike adopts the open conformation (Starr et al., 2021; Dussupt et al., 2021). In comparison, the other two groups of RBD epitopes are accessible to neutralizing antibodies when the spike adopts either open or closed conformation (Scheid et al., 2021; Fedry et al., 2021). In addition to the above conventional antibodies, neutralizing nanobodies (which are single-domain antibodies derived from camelid animals) have also been discovered to target the RBD. Because of their single-domain structure, nanobodies are known to bind to cryptic epitopes on viral targets. We recently discovered four RBD-targeting nanobodies, named Nanosota-1, -2, -3, and -4 (Ye et al., 2021; Ye et al., 2023; Figure 4B). Among them, Nanosota-2 binds to an epitope on the RBD that almost completely overlaps with the ACE2-binding site. Interestingly, the Nanosota-2 epitope is only accessible to Nanosota-2 when the RBD is in the open conformation (Ye et al., 2023). This result suggests that even with the small size of nanobodies, SARS-CoV-2 may be able to evade some of them when its spike is in the closed conformation. Overall, these epitope-based analyses reveal that SARS-CoV-2 spike in the closed conformation can evade significant amounts of neutralizing antibodies, confirming our previous hypothesis that the closed conformation of the spike is a viral strategy for immune evasion (Shang et al., 2020b).

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To further understand the role of the K417 mutation in ACE2 binding and viral entry, we examined how the K417 mutation directly affects the RBD’s binding affinity for ACE2 instead of the trimeric spike’s. The structure of SARS-CoV-2 RBD complexed with human ACE2 showed that Lys417 in SARS-CoV-2 RBD is directly involved in ACE2 binding by forming a salt bridge with Asp30 in human ACE2 (Lan et al., 2020; Figure 5A). In contrast, the corresponding residue in SARS-CoV-1 RBD is a valine that does not form any direct interactions with human ACE2 (Li et al., 2005; Figure 5B). Here, we introduced the K417V mutation to SARS-CoV-2 RBD and investigated the binding interactions between the recombinant SARS-CoV-2 RBD (containing either Lys417 or Val417) and cell-surface-anchored human ACE2 using flow cytometry. The result showed that the K417V mutation reduced the RBD’s binding affinity for ACE2 (Figure 5C, Figure 5—figure supplement 1). Thus, the K417V mutation has contrasting effects on the RBD and the trimeric spike in terms of ACE2 binding: it weakens the RBD’s ability to bind to ACE2 by eliminating a favorable interaction at the RBD/ACE2 interface, but it increases the trimeric spike’s probability of binding to ACE2 by enabling more spike molecules to assume the open conformation.

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Numerical data for Figure 5C.

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To summarize, we identified and investigated a molecular switch regulating the conformation of SARS-CoV-2 spike protein. We used multiple approaches: cryo-EM study of recombinant spike ectodomains, biochemical investigation of cell-surface-anchored full-length and wild-type spikes, spike-mediated pseudovirus entry, and structural analysis of neutralizing antibody/nanobody epitopes. To date, several other studies also investigated the conformation of SARS-CoV-2 spike using cryo-EM (Wrobel et al., 2020; Walls et al., 2020; Ke et al., 2020; Xiong et al., 2020; Cai et al., 2020; Benton et al., 2021), some of which gave different ratios of open and closed spikes probably due to differences in sample preparations and/or protein constructions. In this study, the two spike constructs only differ at residue 417 and the two spike samples were prepared using the same procedure. Importantly, our cryo-EM analysis is consistent with our extensive biochemical and functional approaches. These varied experimental methods complement one another, making this study one of the most thorough in examining the conformation of SARS-CoV-2 spike.

We previously described a unique feature of the prototypic SARS-CoV-2 spike protein: it is present as a mixture of open and closed conformations (Shang et al., 2020b). Building on this discovery, this study demonstrates that the conformation of SARS-CoV-2 spike is regulated, at least in part, by a structural switch. Additionally, this research sheds light on the roles of the conformational changes in the spike’s functions. These findings have implications for the structure, function, and evolution of coronavirus spikes, as well as for the present and potential future coronavirus infections.

How has SARS-CoV-2 spike evolved to reach a balance of open and closed conformations? Through comparative studies of SARS-CoV-2 and SARS-CoV-1 spikes, we identified a molecular switch, RBD residue 417, that regulates the conformational changes of SARS-CoV-2 spike. Lys417 in the closed SARS-CoV-2 spike forms a hydrogen bond with another spike subunit, stabilizing the closed spike. In contrast, Val417 in SARS-CoV-1 spike cannot form such a hydrogen bond with another spike subunit, unable to stabilize the closed spike. When we introduced the K417V mutation to SARS-CoV-2 spike, more spike molecules turned open. Interestingly, Lys417 in the RBD of SARS-CoV-2 spike also directly interacts with ACE2, and we showed that the K417V mutation reduced the RBD’s binding affinity for ACE2. Hence, the K417V mutation has opposing effects on the RBD and the spike for their binding to ACE2. The net outcomes of the K417 mutation are to enhance the spike’s overall binding to ACE2 and the spike’s capability to mediate viral entry.

Besides residue 417, there are likely other molecular factors that influence the opening and closing of SARS-CoV-2 spike. Studies have shown that N-linked glycans on the spike and fatty acids bound to the spike both play a role in regulating its conformation (Toelzer et al., 2020; Sztain et al., 2021). As for protein-based factors, a D614G mutation that emerged later in the pandemic caused SARS-CoV-2 spike to favor the open conformation (Yurkovetskiy et al., 2020; Ozono et al., 2021). Furthermore, our earlier research indicated that three lysine residues kept SARS-CoV-2 Omicron spike in the open conformation (Ye et al., 2022). It is worth noting that residue 417 itself has been a mutational hotspot in SARS-CoV-2 spike; in some pre-Omicron variants, it changed to either asparagine or threonine, and in most Omicron subvariants, it became asparagine (Figure 1C). Given that the side chains of Asn417 and Thr417 might be too short to form a direct interaction with another spike subunit, the mutations at residue 417 could have influenced the conformation of the spike in subsequent SARS-CoV-2 variants. Nevertheless, this study focuses on prototypic SARS-CoV-2, offering comprehensive structural, biochemical, and functional data that pinpoint residue 417 as a key determinant in regulating the conformation of the prototypic spike. The molecular switch at residue 417 could have been a factor in the onset of the COVID-19 pandemic, as discussed below.

How does the conformation of SARS-CoV-2 spike influence both viral entry and immune evasiveness? Our study shows that, compared to its closed form, the open spike is more effective at binding to ACE2 and facilitating viral entry. This could enhance SARS-CoV-2’s ability to infect and spread among humans. We also found that certain epitopes on the RBD are accessible only in the open spike, allowing them to be targeted by neutralizing conventional antibodies and even compact single-domain nanobodies. This suggests the virus can efficiently evade hosts' immune defenses. The virus' ability to switch between open and closed spike conformations may be key to its balance of infectiousness and evasion of immunity. This delicate balance might explain the often mild symptoms and subdued, delayed immune reactions observed in SARS-CoV-2 carriers. In comparison, SARS-CoV-1 spike predominantly assumes the open conformation (89% open and 11% closed) (Gui et al., 2017), potentially leading to more pronounced symptoms and quicker, stronger immune reactions in those infected. Conversely, NL63-CoV spike remains closed (100% closed) (Walls et al., 2016), possibly resulting in even milder symptoms and more muted immune responses. Therefore, SARS-CoV-2’s blend of infectiousness and evasive abilities, differing from both the highly pathogenic SARS-CoV-1 and the milder NL63-CoV, might be a factor in its widespread transmission.

To summarize, Lys417 in SARS-CoV-2 spike aids in keeping the spike closed, potentially hindering ACE2 binding and viral entry, but providing an edge in immune evasion. Simultaneously, Lys417’s direct interaction with human ACE2 boosts ACE2 binding and viral entry, counteracting the limitations of the closed spike. This interesting mechanism helps SARS-CoV-2 maintain a delicate balance between infectiousness and evading host immune response.

All data generated or analysed during this study are included in the manuscript and supporting files. Source data files have been provided for Figures 3 and 5. The atomic models and corresponding cryo-EM density maps have been deposited into the PDB and the Electron Microscopy Data Bank, respectively, with accession numbers PDB 8UUL and EMD-42589 (prototypic SARS-CoV-2 spike containing Lys417 in the closed conformation), PDB 8UUM and EMD-42590 (prototypic SARS-CoV-2 spike containing Lys417 in the open conformation), PDB 8UUN and EMD-42591 (prototypic SARS-CoV-2 spike containing Val417 in the closed conformation), and PDB 8UUO and EMD-42592 (prototypic SARS-CoV-2 spike containing Val417 in the open conformation).

1. 1. Li F

   (2023) RCSB Protein Data Bank

   ID 8UUL. Prototypic SARS-CoV-2 spike containing Lys417 in the closed conformation.

   https://www.rcsb.org/structure/8UUL
2. 1. Li F

   (2023) Electron Microscopy Data Bank

   ID EMD-42589. Prototypic SARS-CoV-2 spike containing Lys417 in the closed conformation.

   https://www.ebi.ac.uk/emdb/EMD-42589
3. 1. Li F

   (2023) RCSB Protein Data Bank

   ID 8UUM. Prototypic SARS-CoV-2 spike containing Lys417 in the open conformation.

   https://www.rcsb.org/structure/8UUM
4. 1. Li F

   (2023) Electron Microscopy Data Bank

   ID EMD-42590. Prototypic SARS-CoV-2 spike containing Lys417 in the open conformation.

   https://www.ebi.ac.uk/emdb/EMD-42590
5. 1. Li F

   (2023) RCSB Protein Data Bank

   ID 8UUN. Prototypic SARS-CoV-2 spike containing Val417 in the closed conformation.

   https://www.rcsb.org/structure/8UUN
6. 1. Li F

   (2023) Electron Microscopy Data Bank

   ID EMD-42591. Prototypic SARS-CoV-2 spike containing Val417 in the closed conformation.

   https://www.ebi.ac.uk/emdb/EMD-42591
7. 1. Li F

   (2023) RCSB Protein Data Bank

   ID 8UUO. Prototypic SARS-CoV-2 spike containing Val417 in the open conformation.

   https://www.rcsb.org/structure/8UUO
8. 1. Li F

   (2023) Electron Microscopy Data Bank

   ID EMD-42592. Prototypic SARS-CoV-2 spike containing Val417 in the open conformation.

   https://www.ebi.ac.uk/emdb/EMD-42592

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Author details

1. Qibin Geng

   1. Department of Pharmacology, University of Minnesota Medical School, Minneapolis, United States
   2. Center for Coronavirus Research, University of Minnesota, Minneapolis, United States

   Contribution

   Conceptualization, Data curation, Formal analysis, Validation, Investigation, Visualization, Writing – review and editing

   Contributed equally with

   Yushun Wan

   Competing interests

   No competing interests declared

2. Yushun Wan

   1. Department of Pharmacology, University of Minnesota Medical School, Minneapolis, United States
   2. Center for Coronavirus Research, University of Minnesota, Minneapolis, United States

   Contribution

   Conceptualization, Data curation, Formal analysis, Validation, Investigation, Visualization, Writing – review and editing

   Contributed equally with

   Qibin Geng

   Competing interests

   No competing interests declared

3. Fu-Chun Hsueh

   1. Department of Pharmacology, University of Minnesota Medical School, Minneapolis, United States
   2. Center for Coronavirus Research, University of Minnesota, Minneapolis, United States

   Contribution

   Data curation, Validation, Investigation, Writing – review and editing

   Competing interests

   No competing interests declared

4. Jian Shang

   1. Department of Pharmacology, University of Minnesota Medical School, Minneapolis, United States
   2. Center for Coronavirus Research, University of Minnesota, Minneapolis, United States

   Contribution

   Data curation, Formal analysis, Validation, Investigation, Writing – review and editing

   Competing interests

   No competing interests declared

5. Gang Ye

   1. Department of Pharmacology, University of Minnesota Medical School, Minneapolis, United States
   2. Center for Coronavirus Research, University of Minnesota, Minneapolis, United States

   Contribution

   Data curation, Formal analysis, Validation, Investigation, Writing – review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-6034-2174

6. Fan Bu

   1. Department of Pharmacology, University of Minnesota Medical School, Minneapolis, United States
   2. Center for Coronavirus Research, University of Minnesota, Minneapolis, United States

   Contribution

   Formal analysis, Validation, Visualization, Writing – review and editing

   Competing interests

   No competing interests declared

7. Morgan Herbst

   1. Department of Pharmacology, University of Minnesota Medical School, Minneapolis, United States
   2. Center for Coronavirus Research, University of Minnesota, Minneapolis, United States

   Contribution

   Data curation, Investigation, Writing – review and editing

   Competing interests

   No competing interests declared

8. Rowan Wilkens

   1. Department of Pharmacology, University of Minnesota Medical School, Minneapolis, United States
   2. Center for Coronavirus Research, University of Minnesota, Minneapolis, United States

   Contribution

   Data curation, Investigation, Writing – review and editing

   Competing interests

   No competing interests declared

9. Bin Liu

   Hormel Institute, University of Minnesota, Austin, United States

   Contribution

   Conceptualization, Resources, Data curation, Formal analysis, Funding acquisition, Validation, Investigation, Visualization, Writing – review and editing

   For correspondence

   liu00794@umn.edu

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-6581-780X

10. Fang Li

    1. Department of Pharmacology, University of Minnesota Medical School, Minneapolis, United States
    2. Center for Coronavirus Research, University of Minnesota, Minneapolis, United States

    Contribution

    Conceptualization, Formal analysis, Supervision, Funding acquisition, Validation, Investigation, Visualization, Writing – original draft, Project administration, Writing – review and editing

    For correspondence

    lifang@umn.edu

    Competing interests

    No competing interests declared

    "This ORCID iD identifies the author of this article:" 0000-0002-1958-366X

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