Structural dynamics in the evolution of SARS-CoV-2 spike glycoprotein

Abstract

SARS-CoV-2 spike glycoprotein mediates receptor binding and subsequent membrane fusion. It exists in a range of conformations, including a closed state unable to bind the ACE2 receptor, and an open state that does so but displays more exposed antigenic surface. Spikes of variants of concern (VOCs) acquired amino acid changes linked to increased virulence and immune evasion. Here, using HDX-MS, we identified changes in spike dynamics that we associate with the transition from closed to open conformations, to ACE2 binding, and to specific mutations in VOCs. We show that the RBD-associated subdomain plays a role in spike opening, whereas the NTD acts as a hotspot of conformational divergence of VOC spikes driving immune evasion. Alpha, beta and delta spikes assume predominantly open conformations and ACE2 binding increases the dynamics of their core helices, priming spikes for fusion. Conversely, substitutions in omicron spike lead to predominantly closed conformations, presumably enabling it to escape antibodies. At the same time, its core helices show characteristics of being pre-primed for fusion even in the absence of ACE2. These data inform on SARS-CoV-2 evolution and omicron variant emergence.

Fig. 1. Differences in structural dynamics between Wuhan (D614) spike and G614 spike.

a A single monomer in closed (top) and open (down) conformation is represented (PDB: 7bnn), with individual domains of spike indicated. b Regions with differences in HDX are superimposed on the structure of G614 spike with one RBD erected (PDB: 7bnn), coloured in red for increased dynamics and in blue for reduced dynamics. Regions not mapped in the structure and showing HDX effects are inserted as dashed lines in one single protomer. c Uptake plots of model peptides of the allosteric axis (FPPR – RBD-s – RBD) that regulates the closed-to-open state transition. d Differential plot illustrates the difference in HDX (ΔHDX) between G614 spike and Wuhan spike across the time points studied. The dashed red light indicates the threshold of significance for increased HDX; the dashed blue line indicates the threshold of significance for reduced HDX. The spike domains are illustrated in the top bar and the green sphere is positioned at the level of the D614G substitution.

Fig. 2. Conformational evolution of spike.

a Map of HDX effects. Regions of various spike trimers with a significant difference in HDX compared to G614 spike are coloured along the spike sequence. In red: increased HDX; in blue: decreased HDX; in magenta: mixed effect; in light grey: no difference in HDX; in dark grey: no information; green spheres: sites of amino acid changes. Yellow rectangles frames regions reporting on a modulation of closed/open state. Orange rectangles frames regions of HDX effects in common among the various spike trimers. b HDX effects are superimposed on the structure of G614 spike with two erect RBDs (PDB: 7bno) for alpha, beta and delta spikes, and on the structure of D614 spike in closed conformation (PDB: 6zge) for omicron spike. Regions not mapped in the structure and showing HDX effects are inserted as dashed lines in one single protomer. Sites with amino acid changes are coloured in green. In the central panel, conformational effects in common among spike variants are superimposed on the NTD subdomain.

Fig. 3. Effect of spike binding on ACE2 dynamics.

a Regions of ACE2 manifesting a significant decrease in HDX upon spike binding are superimposed on the structure of ACE2 ectodomain bound to RBD (PDB: 2ajf), coloured in blue scale according to the magnitude of the HDX effect. The region coloured in red indicates increased HDX upon binding, in dark grey regions with no coverage. b ACE2 binding avidity. The cumulative difference in HDX (ΔHDX) between ACE2 alone and ACE2 bound to spikes and to the isolated ancestral RBD for selected peptides spanning binding sites and across time points 20 s on ice, 10 min at 23 °C and 360 min at 28 °C is plotted. A plot for all time points in Supplementary Fig. 28.

Fig. 4. Effect of ACE2 binding on spikes dynamics.

Regions manifesting decreased HDX in spikes when in complex with ACE2 are superimposed on one single protomer of spike D614 with one RBD bound (PDB: 7a95). In blue: decreased HDX for direct binding in the RBD; in purple: marked decreased HDX in the RBD-s; in cyan: minor decrease in HDX in the RBD-s; sites of amino acid changes in the RBD are indicated with green spheres.

Fig. 5. Cooperative binding (bimodal HDX in the RBM of spikes in complex with ACE2).

The bubble plots of model peptides of the RBM provide an estimation of the level of deuteration and the relative intensity of the two spike subpopulations upon receptor binding. Data are indicative and semi-quantitative and need to be regarded as trends. The bubbles of the low- and high-mass population (bimodal distributions) are sized according to their approximated relative intensity and coloured in blue and red respectively. Magenta bubbles indicate unimodal isotopic distributions, meaning that the two populations converge, uptaking the same level of deuterium. Peptides considered: YGFQPTNGVGYQPYRVVVL (495–513) of Wuhan spike; YGFQPTNGVGYQPYRVVVL (495–513) of G614 spike; YGFQPTNGVGYQPYRVVVL (493–511) of delta spike; YGFQPTYGVGYQPYRVVVL (492–510) of alpha spike; YGFQPTYGVGYQPYRVVVL (492–510) of beta spike; YSFRPTYGVGHQPYRVVV (492–509) of omicron spike.

Fig. 6. Spike priming for fusion.

a Regions manifesting increased HDX in spikes bound to ACE2 are superimposed and coloured in red on a single protomer of the structure of D614 spike with one RBD bound (PDB: 7a95); regions with increased HDX only in spike of alpha and omicron variants bound to ACE2 are coloured in magenta. b The magnitude of the destabilization of the core helices is represented by differential colouring (red scale) for the various spike trimers. The HR1 region (962–982) manifesting HDX bimodality is framed in grey. c The bimodal isotopic envelopes of a model peptide of the HR1 region 962–982 are shown at 15 s and 1 min time points for spike (S) variants in the ACE2-bound state and for omicron spike alone, to exemplify the priming for fusion upon receptor engagement. Bimodal envelopes manifest in omicron spike also in the absence of ACE2, indicating it as pre-primed for fusion.

Fig. 7. Schematic of the structural transition of prefusion spikes.

a Preference for the open state of spikes: Wuhan and omicron spikes favour the closed state, whereas alpha, beta and delta spikes favour the open state. b Priming of spikes upon binding to the ACE2 receptor: alpha, beta and delta spikes bind ACE2 with higher avidity and prime for fusion better than Wuhan spike; omicron spike is primed for fusion better than the other variant spikes and with minimal receptor binding. c Energy landscape of spike in prefusion state. Spikes of alpha and beta variants have more dynamic core helices (higher internal energy) than G614 spike, enhancing their readiness for fusion. Omicron spike presents the most dynamic core helices among all spike variants, which highly increases its internal energy and makes it pre-primed for fusion in the absence of ACE2.