Main

The ongoing global pandemic of the novel SARS-CoV-2 coronavirus presents an urgent need for the development of effective preventative and treatment therapies. The viral spike (S)-protein is a prime target for such therapies because of its critical role in the virus life cycle. The transmembrane CoV S-protein spike trimer is composed of interwoven protomers that include an N-terminal receptor-binding S1 subunit and a C-terminal S2 subunit that contains the fusion elements (Fig. 1a,b)1. The S1 subunit is subdivided into the N-terminal domain (NTD) followed by the receptor-binding domain (RBD) and two structurally conserved subdomains (SD1 and SD2). Together, these domains cap the S2 subunit, protecting the conserved fusion machinery. Binding to the host receptor via the RBD in S1 is followed by proteolytic cleavage of the spike by host proteases2. Large conformational changes in the S-protein result in S1 shedding and exposure of the fusion machinery in S2. Class I fusion proteins, such as the CoV-2 S-protein, undergo large conformational changes during the fusion process and must, by necessity, be highly flexible and dynamic. Indeed, cryo-EM structures of the SARS-CoV-2 spike reveal considerable flexibility and dynamics in the S1 subunit1,2, especially around the RBD, which exhibits two discrete conformational states—a ‘down’ state that is shielded from receptor binding and an ‘up’ state that is receptor-accessible.

Fig. 1: Vector-based analysis of the CoV S-protein demonstrates remarkable variability in the S-protein conformation within ‘up’ and ‘down’ states between CoV strains.
figure 1

a, Cartoon representations of the down (top left) and up (top right) state SARS-2 structures (PDB 6VXX and 6VYB, respectively), colored according to the specified domains (bottom). b, A single down-state protomer of the CoV S-protein with labeled domains (PDB 6VXX). The RBD is in its down conformation. c, A simplified diagram of the CoV S-protein depicting the centroids and vectors connecting them, with the determined angles (θ) and dihedrals (ɸ) labeled. d, The SARS-2 (left, PDB 6VXX) and MERS (right, PDB 6Q04) structures, each with a single protomer depicted in cartoon representation and the remaining two in surface representation. The structures were aligned with the images captured from the same angle for visualization. e, Principal components analysis (PCA) of the SARS and MERS protomers showing measures between the S1 and S2 domains. f, PCA of the SARS, MERS, HKU1 and murine CoV protomers showing measures only between the S1 domains. g, Angle between the SD2-to-SD1 vector and the SD1-to-RBD vector. h, Dihedral about the SD1-to-RBD vector. i, Dihedral about the NTD-to-NTD′ vector. The OC43 value of −162° has been adjusted to 197° (+360°) for visualization. j, Dihedral about the SD2-to-SD1 vector. Data points for SARS, MERS and SARS-2 in g–j are colored according to the up (dark) and down (light) states and the color code in the PCA analysis in e and f. Lines show mean and s.d. The PDB IDs for all structures represented in the PCA and angle/dihedral plots are listed in Supplementary Table 1. k, Structural representation of the g–j angles and dihedrals overlaid on an alignment between a SARS-2 down (cartoon structure with black centroids and lines; PDB 6VXX) and an up (ribbon structure with red centroids and lines; PDB 6VYB) state protomer. Adjacent protomers are depicted as a transparent surface with S1 (light blue) and S2 (light pink). Source data for graphs are provided in Supplementary Data 1.

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The wealth of structural information for β-CoV S-proteins, including the recently determined cryo-EM structures of the SARS-CoV-2 spike1,2,3,4,5,6,7,8,9,10,11, has provided a rich source of detailed geometric information from which to begin precise examination of the macromolecular transitions underlying triggering of this fusion machine. Several structures of soluble ectodomain constructs that retain the complete S1 subunit and the surface-exposed S2 subunit have been determined. These include SARS-CoV-21,3, SARS4,5,6,7,8, MERS4,9 and other human1,10 and murine11 β-CoV S-proteins. These structures revealed remarkable conformational heterogeneity in the S-protein spikes, especially in the RBD region. Within a single protomer, the RBD could adopt a closed down state in which the RBD covers the apical region of the S2 protein near the C terminus of the first heptad repeat (HR1), or an open up state in which the RBD is dissociated from the apical central axis of S2 and the NTD (Fig. 1a). Furthermore, the cryo-EM structures strongly suggest a large degree of domain flexibility in both the down and up states in the NTD and RBD. Although these structures have provided essential information to identify the relative arrangement of these domains, the degree to which conformational heterogeneity may be altered via mutation during the natural evolution of the virus and in a vaccine immunogen design context remains to be determined.

In this study we quantify the variability in the S1 and S2 geometric arrangements to reveal important regions of flexibility to consider and to target for structure-based immunogen design. Based on these analyses, we design mutations that alter the conformational distribution of the domains in the S-protein. We visualize the effect of our designs using a structural determination pipeline relying first on single-particle analysis by negative-stain electron microscopy (NSEM) for rapid and low-cost assessment of the spike ectodomains at low resolution, followed by cryo-EM for high-resolution information on the changes introduced by these mutations. Our results reveal a heterogeneous conformational landscape of the SARS-CoV-2 spike that is highly susceptible to modification by the introduction of mutations at sites of contact between the S1 and S2 subunits. We also present data on modified SARS-CoV-2 ectodomain constructs stabilized in conformations that have not yet been seen in the current available structures, with great interest and direct application in vaccine design.

Results

Defining domain geometry and arrangements in SARS-CoV-2 spike

To characterize the unique arrangement of distinct domains in the different β-CoV spikes, we first developed a quantitative definition of their relative positions (Fig. 1). Examination of available SARS and MERS S-protein structures revealed the following: (1) domains in the S1 subunit (NTD, RBD and subdomains) and in the S2 subunit (connector domain (CD)) move as rigid bodies and (2) these domains display a pronounced array of relative shifts between those in the S1 subunit and the S2 subunit β-sheet motif and CD. To quantify these movements, we analyzed the relevant regions of motion and their structural disposition in available β-CoV ectodomain spike structures, including 15 for SARS4,5,7,8, 16 for MERS4,8,9,12, one each for HKU11,10, OC431,10 and a murine β-CoV, and three SARS-CoV-23,13 structures (Fig. 1e–j and Extended Data Fig. 1). Each protomer in these structures displaying asymmetric up/down RBD states was examined independently, yielding a dataset of 83 structural states. Structures lacking the RBD were not analyzed. Only the S1 subunits were analyzed for the HKU1, OC43 and murine β-CoV S-protein structures. Information regarding whether the constructs are cleaved, mutated and/or liganded is provided in Supplementary Table 1.

The NTD was split into a primary N-terminal section and a secondary C-terminal section based on visual inspection of this region in the various β-CoV structures. Vectors connecting each region’s Cα centroids were generated and used to define the relative dispositions of the domains (Fig. 1b,c). The vector magnitudes and select angles and dihedrals were used to identify the breadth of differences in domain positioning and compare between β-CoV viruses (Fig. 1e–j). Projecting the S1 and S2 subunit vector data for SARS-CoV-1, MERS and SARS-CoV-2 using the principal components analysis method revealed that each virus’s S-protein structures resided in distinct clusters (Fig. 1e). A similar analysis of the S1 subunit vectors with the addition of data from the murine β-CoV, HKU1 and OC43 generally retained these clusters, with the three additional structures lying in relatively unique positions (Fig. 1f). The SARS-CoV-1 structures split into two distinct clusters, separated largely by differences in the up/down states, with SARS-CoV-2 structures splitting with it. The distinct clustering of SARS-CoV-1 compared to SARS-CoV-2 in the PCA results, which include the S2 subunit data, suggests that there are key differences in the S1 disposition relative to S2 between SARS-CoV-1 and SARS-CoV-2.

Consistent with the PCA analysis, examination of specific angles and dihedrals indicated that β-CoV S-proteins in various viruses differ markedly from one another (Fig. 1g–j). This is in addition to considerable variability in the domain arrangements within different virus S-proteins, demonstrating its wide structural lability (Fig. 1e–j). In particular, both ɸ1 and ɸ3 (Fig. 1h,j), describing the dihedrals about the vector connecting SD2 to SD1 and the vector connecting SD1 to the RBD (Fig. 1b,c), respectively, effectively reported on the up and down configurations and indicated substantial differences between SARS-CoV-1 and MERS in both the up and down states. Differences in the angular disposition of the NTD elements of SARS-CoV-1 and MERS as measured by ɸ1, θ2 and θ4 were also observed (Fig. 1i and Extended Data Fig. 1a,c). Additional S1 and S1/S2 subunit differences between viruses were observed for vectors involving SD2. This includes the SD2 to CD ɸ4 dihedral, which differs markedly between MERS/SARS-CoV-2 and SARS-CoV-1, and the angle between the vectors connecting the NTD′ to SD2 and SD2 to the CD, demonstrating a shift in SARS-CoV-2 (Extended Data Fig. 1e,g). Finally, the disposition of the CD to the inner portion of S2 measured as an angle between a vector connected to an interior S2 β-sheet motif and the vector connecting the CD to SD2 indicates SARS-CoV-1 differs from both MERS and SARS-CoV-2 (Extended Data Fig. 1f). The MERS disposition appears to respond to RBD triggering in some structures, displaying a bimodal distribution (Extended Data Fig. 1f).

These results demonstrate that, although the individual domain architectures and overall arrangements are conserved, important differences between these domains exist between viruses, suggesting that subtle differences in interdomain contacts could play a major role in determining these distributions and thereby alter surface antigenicity and the propensity of the domains to access up and down RBD states.

Differential stabilization of SARS-CoV-2 spike ectodomain RBD orientation

Based on the observed variability in the analysis of β-CoV spikes, we asked whether the propensity for the RBD to display the down and up states could be modified via mutations, without altering the exposed antigenic surfaces. We focused on domain pairs that moved relative to each other (Fig. 1) and identified interaction sites that could alter the S-protein conformational distribution. That is, modification of the up/down equilibrium could be achieved by modifying either the RBD or subdomain contact surfaces. To this end, we selected contact regions between the RBD and S2, the RBD and NTD, SD1 and S2, and SD2 and S2. Mutations at these sites were selected based on in silico mutagenesis using the Schrödinger Biologics suite14,15,16. To eliminate exposure of the receptor binding site of the RBD, we examined the potential for disulfide linkages between the RBD and its contact with S2 near the C terminus of HR1 to prevent RBD exposure.

We identified a double cysteine mutant, S383C D985C (RBD to S2 double mutant (rS2d), Extended Data Fig. 2), as a candidate. The transition from the down state to the up state involves shifts in the RBD to NTD contacts. Therefore, to prevent these shifts, we identified a site in an RBD groove adjacent to the NTD, and prepared a triple mutant, D398L S514L E516L (RBD to NTD triple mutant (rNt), Extended Data Fig. 2). As SD1 acts as a hinge point for the RBD up/down transitions (Figs. 1a–c and 2i,j), we hypothesized that enhanced hydrophobicity at the SD1 to S2 interface might shift the position of SD1, thus influencing the hinge and potentially the propensity for RBD triggering. A double mutant, N866I A570L (subdomain 1 to S2 double mutant (u1S2d)), and a quadruple mutant, A570L T572I F855Y N856I (subdomain 1 to S2 quadruple mutant (u1S2q)), were identified for this purpose (Extended Data Fig. 2). Finally, we identified a double cysteine mutant, G669C and T866C, to link SD2 to S2 (subdomain 2 to S2 double mutant (u2S2d), Extended Data Fig. 2).

Fig. 2: Cryo-EM structures reveal differential stabilization of the S-protein in the mutant ectodomain constructs.
figure 2

a, The S-protein spike, highlighting the two regions of interest for structure and computation-based design (PDB 6VXX). b, The rS2d RBD-to-S2 locked structure displaying only the all-RBD-down state map. c, The u1S2q SD1-to-S2 mutated structure displaying the all-RBD-down state, the 1-RBD up state and the 2-RBD up state. The percentage shown in orange is the sum of particles found in either the 1- or 2-RBD up state. Each protomer in each map is colored differently, with resolutions for these listed below. Red asterisks indicate up-state RBDs.

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NSEM analysis of SARS-CoV-2 spike ectodomain proteins

The mutants were prepared in the context of a previously published SARS-CoV-2 ectodomain construct3 (Extended Data Fig. 3). To assess the quality of the purified proteins, we performed NSEM analysis (Extended Data Fig. 4). The micrographs show a reasonably uniform distribution of particles consistent with the size and shape of the SARS-CoV-2 spike ectodomain. Two-dimensional (2D) class averages showed spike populations with well-resolved domain features.

Following 3D classification then homogeneous refinement, the unmutated spike resolved into two classes of roughly equal proportions that differed in the position of their RBD domains. One class displayed all three RBDs in their down positions, whereas the other class displayed one RBD in the up position. This was consistent with published cryo-EM results13 that describe a 1:1 ratio between the down and 1-up states of the SARS-CoV-2 spike ectodomain.

The mutant spikes, analyzed using a similar workflow, showed different conformation distributions compared to the unmutated spike. The most pronounced changes were observed in the rS2d and u1S2q constructs (Extended Data Fig. 4c,e). For the rS2d construct, we observed only the down conformation (Extended Data Fig. 4c); the 1-RBD up state was not found in this dataset. In the u1S2q data, we observed an S-protein population with 2 RBDs in the up position, which has been reported before for the SARS-CoV-1 and MERS CoV spike ectodomains7,9. These two constructs were selected for downstream, high-resolution characterization by cryo-EM.

Cryo-EM and properties of the SARS-CoV-2 spike constructs

We collected cryo-EM datasets for the rS2d and u1S2q constructs (Figs. 2–6, Table 1 and Extended Data Figs. 5–9). Consistent with the NSEM analysis, following multiple rounds of 2D and 3D classification, we found a population of down state spike in the rS2d dataset (Fig. 3 and Extended Data Fig. 6). We then implemented additional exhaustive ab initio classifications, as well as heterogeneous classifications using low-pass filtered maps of known open conformations of CoV spikes to search for open state spikes in the dataset. We were unable to find any such states, suggesting that the disulfide bond between the RBD and the S2 subunit effectively locked the SARS-CoV-2 spike in its down conformation. The rS2d cryo-EM reconstruction had an overall resolution of 2.7 Å, but the local resolution varied widely (Extended Data Fig. 6g), with the highest resolutions in the S2 subunit (Extended Data Fig. 6h) and higher disorder and lower local resolutions in the S1 subunit; this is consistent with previous structures of the SARS-CoV-2 spike3,13. We observed connecting density at the site of the engineered disulfide link in rS2d, thus confirming disulfide formation (Fig. 3c–e). Alignment of this structure with that of the unmutated down closed state structure (PDB 6VXX)13 indicated that the overall protein structure was otherwise unperturbed (root-mean-square deviation (r.m.s.d.) of 0.5 Å).

Fig. 3: Cryo-EM structure of the rS2d construct locked in the down state.
figure 3

a, Cryo-EM reconstruction of rS2d, colored by chain. b, Rotated view (by ~90°) of a. c, Magnified view showing the engineered disulfide linking the RBD of one protomer and the S2 domain of the adjacent protomer. The disulfide bridge is shown as spheres and colored by elements. d, Rotated view (by ~90°) of c. e, Magnified view of the engineered interchain disulfide showing the cryo-EM density as a transparent surface and the underlying model in stick representation. f, Binding of antibody CR3022 to the unmutated structure (black bar) and rS2d (red bar). The spike was captured on a streptavidin chip and binding to CR3022 immunoglobulin-G (IgG) was measured by surface plasmon resonance (SPR). Data shown are means and s.d. of three technical repeats and are representative of five independent experiments.

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Fig. 4: Cryo-EM structures of RBD-down S-proteins reveal differential stabilization of domain positions.
figure 4

a, Cryo-EM reconstruction of u1S2q colored by chain. b, Rotated view (by ~90°) of a. c, Magnified view of the region containing the mutations, showing the proximity of the F855Y and N856I residue loop to the S2 residue L966 and S1 residue P589. d, Similar region to that shown in c but for the unmutated structure (PDB 6VXX). e, Magnified view of the region containing the mutations at a different angle than the one shown in c. f, The loop containing the A570L and T572I mutations, with the cryo-EM map shown as a transparent surface and fitted model shown in cartoon representation. g,h, Similar region to that shown in e but for the rS2d structure (PDB 6X29) (g) and the unmutated structure (PDB 6VXX) (h). i,j, Overlay of the three down state structures (i) and with the cryo-EM reconstruction for the down state for the u1S2q construct (j).

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Fig. 5: Cryo-EM structure of the u1S2q 1-RBD up state reveals increasing relaxation of the triggered RBD toward the unmutated structure.
figure 5

a, Cryo-EM reconstruction of the u1S2q 1-RBD up state colored by chain. b, Rotated view (by ~90°) of a. c–e, Magnified views of the region containing the A570L and T572I mutations in the ‘up’ RBD protomer (c), the ‘up’-coupled ‘down’ RBD protomer (d) and the ‘down’ RBD protomer (e) of the asymmetric 1-RBD up spike. The cryo-EM reconstruction is shown as a transparent surface with the underlying fitted model in cartoon representation and residues as balls and sticks. f–h, The 1-RBD up structure of the unmutated spike (PDB 6VYB) (shown in black and gray) superimposed on the u1S2q 1-RBD up structure (PDB 6X2B, this study), colored according to the coloring scheme in a. Overlays are shown for the ‘up’ RBD protomer (f), the ‘up’-coupled ‘down’ RBD protomer (g) and the ‘down’ RBD protomer (h).

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Fig. 6: Structure of the u1S2q 2-RBD up state.
figure 6

a, Cryo-EM reconstruction of u1S2q, colored by chain. b, Rotated view (by ~90°) of a. c, Binding of antibody CR3022 to the unmutated construct (top) and u1s2q (bottom). Binding of CR3022 Fab to the S-proteins was measured by SPR using single-cycle kinetics. The black lines are the binding sensorgrams and the red lines show fits of the data to a 1:1 Langmuir binding model. d, CR3022 (shown as a semi-transparent, gray surface) modeled on the 1-up u1S2q structure (left) and the 2-up u1S2q structure (right) using RBD in the crystal structure of the CR3022–RBD complex (PDB 6W41) to superimpose on the up RBD of the u1S2q structures. Locations of potential clashes are indicated in each model.

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Table 1 Cryo-EM data collection, refinement and validation statistics
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We next tested the binding of rS2d to antibody CR3022 (Fig. 3f and Extended Data Fig. 7a)17,18. CR3022 binds an epitope on the RBD of the SARS-CoV-1 and SARS-CoV-2 spikes that is occluded in the down RBD conformation. Although we observed robust binding for the unmutated spike to CR3022, no measurable binding was observed for rS2d, confirming that the RBD in the rS2d construct is locked in a down conformation. Also, as expected, the rS2d mutant showed reduced binding to angiotensin-converting enzyme 2 (ACE-2) compared to the unmutated spike (Extended Data Fig. 7b,c), and such activity could be abrogated using an ACE-2 or a CR3022 IgG-immobilized column for purification (Extended Data Fig. 7c–h).

In contrast to rS2d, the u1S2q spike displayed widespread rearrangement of the S1 subunits (Extended Data Figs. 8 and 9). In the down state structure, the mutated S2 position remained in the configuration observed in the unmutated construct, with the F855Y and N856I residue loop in close proximity to S2 residue L966 and S1 residue P589 (Fig. 4c,d), suggesting that these mutations had little impact on the observed shifts. However, the S2-interactive SD1 displayed a rigid body movement relative to both the rS2d and unmutated constructs, with θ1 and ɸ3 displacements of 3.7° and 2.4°, respectively (Fig. 4e–j and Extended Data Fig. 10). This resulted in displacement of the A570L + T572I-containing loop from the unmutated position near the S2 L966 residue (Fig. 4i,j). The S2 contact disruption is accompanied by an angular shift of the NTD away from the primary trimer axis due to SD1 to NTD′ contacts, yielding θ3 and ɸ2 shifts of 6.0° each, respectively (Extended Data Fig. 10). The subdomain rearrangement impacts the positioning of the RBD with only a minor shift in the ɸ1 dihedral of 0.1° indicating that the RBD moved with SD1, indicated in the θ1/ɸ3 shifts. The newly acquired arrangement in both the RBD and NTD was further accompanied by an apparent increase in their flexibility, suggesting conformational heterogeneity. These down state shifts were observed in both the single RBD up structure and the two RBD up structures (Figs. 5 and 6 and Extended Data Fig. 10). The extent to which the SD1 shift differed from that observed in the unmutated construct was context-dependent in the 1-RBD up state (Fig. 5c–h). Although the down state RBD in contact with the up state RBD displayed the large shift in position observed in the all down state (Fig. 5d,g), the down state RBD with its terminal position free displayed an intermediate SD1 configuration (Fig. 5e,h). The up state RBD in the u1S2q construct resided largely in the position occupied in the unmutated construct (Fig. 5c,f). This indicated that the effect of the mutations was primarily isolated to the down state and suggested these mutations act to destabilize the down state rather than to stabilize the up state. These features were largely recapitulated in the u1S2q 2-RBD up state conformation, with subdomain 1 retaining the shift in the down state RBD (Supplementary Table 1). The structural details presented here indicate that, while locking the down state RBD into its unmutated position had little impact on the overall configuration of S1, altering the disposition of SD1 had wide-ranging impacts, consistent with the observed virus-to-virus differences in the geometric analysis described in Fig. 1.

We measured the binding of u1S2q with antibody CR3022 and ACE-2 (Fig. 6c and Extended Data Fig. 7a–c) and observed a modest but reproducible increase in ACE-2 binding for u1S2q compared to the unmutated spike, consistent with the higher propensity of u1S2q to adopt RBD-up conformations (Extended Data Fig. 7b,c). The affinities of the CR3022 antibody to the unmutated spike or u1S2q were similar and also consistent with the previously published affinity of CR3022 for an RBD-only construct17 (Fig. 6c). On docking of the crystal structure of the CR3022 Fab in complex with the RBD (PDB 6YLA)17 onto the 1-up and 2-up structures of u1S2q, we found that the CR3022 constant domain clashed with the NTD and the adjacent RBD in both the 1-up conformation and the 2-up conformations (Fig. 6d,e). As previously suggested17, CR3022 binding is therefore likely to require a conformational change that rotates the RBD away from the central axis of the spike. In summary, none of the structures of the structural states of the spike determined thus far are compatible with CR3022 binding and our data show that CR3022 must induce or capture this potentially transient state in both the unmutated and u1S2q spikes.

Discussion

Conformational plasticity is a hallmark of the enveloped-virus fusion-protein structure due to the necessity of protecting the conserved viral fusion elements from host immune responses while retaining a sufficiently steep free-energy gradient to enable host cell fusion19. Exposed elements are generally well conditioned to be permissive and responsive to mutations through genetic drift and host immune adaptation. Conformational plasticity, however, presents an important difficulty in the context of vaccine and drug design. Indeed, lessons learned in the continued effort to produce a broadly protective HIV-1 vaccine have demonstrated the importance of a detailed understanding and control of fusion protein dynamics20,21,22,23,24,25,26,27,28,29,30,31. SARS-CoV-2 is probably no exception in this regard and, indeed, the conformational plasticity of the SARS-CoV-2 S-protein appears greater than that of the HIV-1 Env.

We aimed to develop a quantitative understanding of β-CoV structural states between viruses and within each RBD down and up state configuration. The wide breadth of domain arrangements and the relatively small contact area between the S1 and S2 subunits suggest that large changes in S-protein structure may occur from few mutations. A recent report indicated that the D614G mutation (located in the SD2 region and contacting S2) causes a potential fitness gain32. Based on our results showing that a disulfide linkage between SD2 and S2 in u2S2d decreased the population of up state RBDs (Extended Data Fig. 4b), the D614G mutation may indeed alter the conformational landscape of the SARS-CoV-2 S-protein. It was previously noted that the RBD up states observed in MERS and SARS-CoV-1 were not observed in the OC43, MHV or HKU1 S-protein structures10. We note that the SD1 u1S2q mutation sites are all asparagine in those other CoV S-proteins, and each displays a marked difference in SD1 positioning relative to the u1S2q structure. Thus, sequence differences at interdomain contact sites are not uncommon, and our observations suggest that the β-coronaviruses can quickly evade conformationally selective antibody responses. However, the degree to which this evasion mechanism is effectively utilized by the virus is uncertain, as such mutations may simultaneously incur a fitness penalty.

From the perspective of immunogen development, the constructs developed here present an opportunity to examine the ability of differentially stabilized S-protein particles to induce two different, yet important antibody responses. First, the u1S2q construct developed here displayed a prominent two-RBD up state distribution, demonstrating that interdomain contact modifications can lead to higher exposure of the immunogenic receptor-binding regions. Because RBD-directed responses that target the ACE-2 binding site form a dominant proportion of neutralizing immune responses in infected patients33, constructs that increase exposure and access to these sites could be useful candidates for vaccination regimens aimed at eliciting such responses. Indeed, full-length SARS-CoV-2 spike structures, either detergent-solubilized34 or virion-associated35, suggest that the down state is the predominant state that, in a vaccination context, may limit RBD-directed responses. The u1S2q mutations may therefore provide a means to enhance the induction of RBD responses in full-length S-protein immunogens. Furthermore, this design could also be used to identify receptor-binding-site-directed antibodies from patient sera, including potential new antibodies that may target sites that require a two-RBD up conformation. Second, the disulfide-linked down state locked double mutant (rS2d) would presumably not elicit antibodies targeting the receptor binding site (these make up the majority of observed responses in convalescent patients36,37), but it would still be capable of eliciting antibodies such as S309 that are able to bind the down state RBD38. Indeed, a study of MERS responses suggests non-RBD responses (particularly NTD and S2 epitopes) will play an important role in vaccine-induced protection39. From a theoretical perspective, the wide control over the RBD up/down distribution available to the virus suggests that, by analogy to HIV-1 viruses, which are notoriously difficult to neutralize, conformational blocking of antibody responses would not be unusual. Although there might be a fitness cost to the virus, it would not necessarily render the virion non-infectious. Using the double mutant rS2d as an immunogen provides a platform for inducing non-RBD responses that may be needed to protect against such evasion. Although the low expression yields of rS2d are a potential hindrance to its utility, introduction of other mutations, such as the recently described mutations designed to stabilize the S2 region40, may provide a way to further stabilize rS2d and increase its utility as an immunogen.

Complicating factors, such as a potential for antibody-induced enhancement, may favor the use of truncated, single-domain constructs with fewer potentially weak or non-neutralizing epitopes. Nevertheless, the designs presented here will allow for a detailed characterization of vaccine immunogenicity and antigenicity, paving the way for the development of vaccines for the novel SARS-CoV-2 and eventually a broadly protective β-CoV vaccine. Thus, although the previous generation of stabilizing mutations ensured well-folded spike trimers, the rational design approach developed here provides a means to controlling the RBD orientation distribution and should allow us to explore the impact of conformational dynamics from the perspective of vaccine and drug development.

Methods

Vector-based analysis

Vector analysis was performed using available cryo-EM structures for SARS-CoV-23,13, SARS4,5,7,8, MERS4,9 and other human1,10 and murine11 β-CoV spike proteins. Domains for the vector analysis were selected based on visual inspection of alignments between SARS, MERS and SARS-CoV-2 structures. Specifically, Cα centroids were determined for the S1 NTD, RBD, SD1 and SD2 (SARS-CoV-2 residues 27–43 and 54–271, 330–443 and 503–528, 323–329 and 529–590, 294–322 and 591–696, respectively; equivalent SARS/MERS/murine/HKU1/OC43 residues selected based on structural alignment with SARS-CoV-2), as well as a β-sheet motif in the NTD (residues 116–129 and 169–172) and a helix motif in the RBD (residues 403–410). The NTD was split into two regions with the SD1-contacting, SD2-adjacent portion referred to here as NTD′ (residues 44–53 and 272–293). Cα centroids in the S2 subunit were obtained for a β-sheet motif (residues 717–727 and 1047–1071) and the CD domain (711–716 and 1072–1122). Vector magnitudes, angles and dihedrals between these centroids were determined and used in the subsequent analysis. Vector analysis was performed using the VMD41 Tcl interface. PCA was performed in R with the vector data centered and scaled42.

Rational structure-based design

Structures for SARS-COV-1 (PDB 5X584), MERS (PDB 6Q0412) and SARS-CoV-2 (PDB 6VXX13) were prepared in Maestro43 using the protein preparation wizard14 followed by in silico mutagenesis using Schrödinger’s cysteine mutation15 and residue scanning16 tools. Residue scanning was first performed for individual selected sites allowing mutations to Leu, Ile, Trp, Tyr and Val, followed by scanning of combinations for those that yielded a negative overall score. Scores and visual inspection were used in the selection of the prepared constructs.

Protein expression and purification

The SARS-CoV-2 ectodomain constructs were produced and purified as described previously3. Briefly, a gene encoding residues 1−1208 of the SARS-CoV-2 S (GenBank MN908947) with proline substitutions at residues 986 and 987, a ‘GSAS’ substitution at the furin cleavage site (residues 682–685), a C-terminal T4 fibritin trimerization motif, an HRV3C protease cleavage site, a TwinStrepTag and an 8XHisTag was synthesized and cloned into the mammalian expression vector pαH. All mutants were introduced in this background. Expression plasmids encoding the ectodomain sequence were used to transiently transfect FreeStyle293F cells using Turbo293 (SpeedBiosystems). Protein was purified on the sixth day post-transfection from the filtered supernatant using StrepTactin resin (IBA).

Antibody CR3022 was produced in Expi293 cells and purified by Protein A affinity. To express the ACE-2 constructs, the ACE-2 gene was cloned as a fusion protein with either the human or mouse Fc region attached to the C-terminal end of ACE-2. A 6X His-tag was added to the C-terminal end of the Fc domain of each construct. ACE-2 with human Fc tag was purified by Protein A affinity chromatography, and ACE-2 with mouse Fc tag was purified by Ni-NTA chromatography.

Thermal shift assay

The thermal shift assay was performed using Tycho NT.6 (NanoTemper Technologies). Spike variants were diluted (0.15 mg ml−1) in nCoV buffer (2 mM Tris, pH 8.0, 200 mM NaCl, 0.02% sodium azide) and run in duplicates in capillary tubes. Intrinsic fluorescence was recorded at 330 nm and 350 nm while heating the sample from 35 to 95 °C at a rate of 3 °C min−1. The ratio of fluorescence (350/330 nm) and the inflection temperatures (Ti) were calculated by Tycho NT.6.

Negative-stain electron microscopy

A 100 µg ml−1 final concentration of the spike was made in 5% glycerol in HBS pH 7.4 (20 mM HEPES, 150 mM NaCl) with 7.5 mM gluteraldehyde at room temperature. After 5-min incubation, gluteraldehyde was quenched by adding sufficient 1 M Tris stock, pH 7.4 to give a final concentration of 75 mM Tris and incubated for 5 min. The carbon-coated grids (CF300-cu, EMS) were glow-discharged for 20 s at 15 mA. A 5-µl sample incubated on grid for 10–15 s was blotted and then stained with 2% uranyl formate. Images were obtained with a Philips EM420 electron microscope operated at 120 kV, at ×82,000 magnification and with a pixel size of 4.02 Å. The RELION44 program was used to perform class averaging of the single-particle images.

Cryo-electron microscopy sample preparation, data collection and processing

Purified SARS-CoV-2 spike preparations were diluted to a concentration of ~1 mg ml−1 in 2 mM Tris pH 8.0, 200 mM NaCl and 0.02% NaN3, then 2.5 µl of protein was deposited on a CF-1.2/1.3 grid that had been glow-discharged for 30 s in a PELCO easiGlow glow discharge cleaning system. After 30-s incubation in >95% humidity, excess protein was blotted away for 2.5 s before being plunge-frozen into liquid ethane using a Leica EM GP2 plunge freezer (Leica Microsystems). Frozen grids were imaged in a Titan Krios system (Thermo Fisher) equipped with a K3 detector (Gatan). Data were acquired using the Leginon system45. The dose was fractionated over 50 raw frames and collected at a 50-ms frame rate. This dataset was energy-filtered with a slit width of 30 eV. Individual frames were aligned and dose-weighted46. CTF estimation, particle picking, 2D classifications, ab initio model generation, heterogeneous refinements, homogeneous 3D refinements and local resolution calculations were carried out in cryoSPARC47.

Cryo-electron microscopy structure fitting and analysis

Structures of the all down state (PDB 6VXX) and single RBD up state (PDB 6VYB) from the previously published SARS-CoV-2 ectodomain were used to fit the cryo-EM maps in Chimera48. The 2-RBD up state was generated in PyMol using the single RBD up state structure. Mutations were made in PyMol49. Coordinates were then fitted manually in Coot50 followed by iterative refinement using Phenix51 real space refinement and subsequent manual coordinate fitting in Coot. Structure and map analysis were performed using PyMol, Chimera48 and ChimeraX52.

Surface plasmon resonance

The binding of antibody CR3022 and ACE-2 to the SARS-CoV-2 spike constructs was assessed by SPR, in two experimental formats, on a Biacore T-200 system (GE Healthcare) at 25 °C using HBS-EP+ (10 mM HEPES, pH 7.4, 150 mM NaCl, 3 mM EDTA and 0.05% surfactant P-20) as the running buffer. In the first format, the spike constructs were captured on an SA chip and were assayed by flowing over CR3022 IgG or ACE-2-Fc. The surface was regenerated between injections by flowing over SA regeneration buffer (1 M NaCl, 50 mM NaOH) solution for 10 s with a flow rate of 100 µl min−1. Blank sensorgrams were obtained by injection of the same volume of HBS-EP+ buffer in place of IgGs and Fab solutions. Sensorgrams were corrected with corresponding blank curves. In the second format, CR3022 IgG or ACE-2-human Fc was captured on CM5 chip immobilized with human Anti-Fc (8,000 RU), and binding was measured by flowing over each of the spike constructs at 200 nM in running buffer. The surface was regenerated between injections by flowing over 3 M MgCl2 solution for 10 s with a flow rate of 100 µl min−1. To assess the affinity of CR3022 to the unmutated and u1S2q spikes, the spikes were captured on an SA chip as described above, and five concentrations of CR3022 were injected in a single-cycle kinetics format. Sensorgram data were analyzed using the BiaEvaluation software (GE Healthcare).

Reporting Summary

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