SARS-CoV-2 Variants Increase Kinetic Stability of Open Spike Conformations as an Evolutionary Strategy

Abstract

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VOCs) harbor mutations in the spike (S) glycoprotein that confer more efficient transmission and dampen the efficacy of COVID-19 vaccines and antibody therapies. S mediates virus entry and is the primary target for antibody responses, with structural studies of soluble S variants revealing an increased propensity toward conformations accessible to the human angiotensin-converting enzyme 2 (hACE2) receptor. However, real-time observations of conformational dynamics that govern the structural equilibriums of the S variants have been lacking. Here, we report single-molecule Förster resonance energy transfer (smFRET) studies of critical mutations observed in VOCs, including D614G and E484K, in the context of virus particles. Investigated variants predominately occupied more open hACE2-accessible conformations, agreeing with previous structures of soluble trimers. Additionally, these S variants exhibited slower transitions in hACE2-accessible/bound states. Our finding of increased S kinetic stability in the open conformation provides a new perspective on SARS-CoV-2 adaptation to the human population. IMPORTANCE SARS-CoV-2 surface S glycoprotein-the target of antibodies and vaccines-is responsible for binding to the cellular receptor hACE2. The interactions between S and hACE2 trigger structural rearrangements of S from closed to open conformations prerequisite for virus entry. Under the selection pressure imposed by adaptation to the human host and increasing vaccinations and convalescent patients, SARS-CoV-2 is evolving and has adopted numerous mutations on S variants. These promote virus spreading and immune evasion, partially by increasing the propensity of S to adopt receptor-binding competent open conformations. Here, we determined a time dimension, using smFRET to delineate the temporal prevalence of distinct structures of S in the context of virus particles. We present the first experimental evidence of decelerated transition dynamics from the open state, revealing increased stability of S open conformations to be part of the SARS-CoV-2 adaption strategies.

Keywords: SARS-CoV-2 variants; conformational dynamics; single-molecule FRET; spike glycoprotein; structure.

FIG 1

Experimental design for characterizing the conformational dynamics of the SARS-CoV-2 spike variants on virus particles via single-molecule FRET. (A and B) Experimental setup. (A) Virus-like particles that carry a single two-dye-labeled SARS-CoV-2 spike protomer among unlabeled wild-type spikes were immobilized on a quartz slide and then imaged on a prism-based total internal reflection fluorescence (TIRF) microscope. The quartz slide was passivated with PEG/PEG-biotin to enable streptavidin coating and the subsequent immobilization of virions that contain a biotin-lipid (DSPE-PEG-biotin). The virus-like particles were composed of HIV-1 cores and SARS-CoV-2 spikes on the virus surface. (B) Positioning of labeling dyes for the site-directed incorporation of fluorophores (LD555-cadaverine [a Cy3B derivative], green; LD655-CoA [a Cy5 derivative], red) was elucidated by the S1 conformational change from the RBD-down to the RBD-up structures, which are adapted from RCSB PDB codes 6ZB5 (all-RBD-down) and 7KJ5 (one-RBD-up). (C) Domain organization of the parental full-length SARS-CoV-2 spike protein with D614G and E484K mutations. The introduction sites of labeling tags Q3 (green) and A4 (red), where the donor and acceptor fluorophores will be conjugated to, respectively, are indicated. SD1, subunit domain 1; SD2, subunit domain 2; S1/S2, S2′, protease cleavage sites; FP, fusion peptide; HR1/HR2, heptad repeat 1/heptad repeat 2; TM, transmembrane domain; CT, cytoplasmic tail. Labeling peptides Q3 and A4 were inserted into RBD and SD1, respectively, before and after the RBM within the S protein in these studies. Emerged variants that are currently classified by CDC as variants of concern and their respective key mutations in the spike are specified in the table.

FIG 2

D614G substitution shifts the conformational landscape of unbound spike from the ground state to asymmetrically configurated intermediate states. (A) Infectivity quantification for HIV-1 lentivirus particles carrying various spike variants determined on hACE2 expressing 293T cells (293T-ACE2). Infectivity (mean ± SD) was measured from three independent experiments in triplicates. RLU, relative light units. (B) Example fluorescence traces (LD555, green; LD655, red) and resulting quantified FRET traces (FRET efficiency, blue; hidden Markov model initialization, red) of a dually labeled ligand-free spike protein on the surface of HIV-1 lentivirus particle. The single-step photobleaching of dyes at the single-molecule level defined the baseline (dashed black). Four distinguishable FRET-populated states are indicated as color-coded dashed lines. (C and D) FRET histograms (left) and TDPs (right) of ligand-free D614 spike (S_D614 [C]) and G614 spike (S_G614 [D]) on lentivirus particles. A number (N_m) of individual active/dynamic molecule-FRET traces were compiled into a conformation-population FRET histogram (gray lines) and fitted into a 4-state Gaussian distribution (solid black) centered at 0.1-FRET (dashed cyan), 0.3-FRET (dashed red), 0.5-FRET (dashed green), and 0.75-FRET (dashed magenta). TDPs show the distributions of initial and final FRET values for every observed transition in FRET traces. TDPs are displayed as initial FRET versus final FRET with relative frequencies (max red scale = 0.01 transitions/second), originated from the idealization of individual FRET traces in FRET histograms. TDPs trace the locations of state-to-state transitions and their relative frequencies. (E and F) Experiments as in panels C and D, respectively, conducted in the presence of 200 μg/mL of monomeric hACE2. The soluble hACE2 activates spike proteins on the virus by shaping the conformational landscape toward stabilizing the all-RBD-up conformation (activated state). FRET histograms represent mean ± SEM, determined from three randomly assigned populations of FRET traces under corresponding experimental conditions. For evaluated relative state occupancies, see Table S1.

FIG 3

E484K stabilizes the S_Alpha variant toward RBD-up conformations. (A and B) FRET histograms (left) and TDPs (right) of S_Alpha variant on lentivirus particles with (A) and without (B) 200 μg/mL of hACE2. The soluble hACE2 activates S_Alpha by shaping the conformational landscape toward stabilizing the all-RBD-up conformation. (C) Representative fluorescence traces (LD555, green; LD655, red) and quantified FRET traces of a single ligand-free E484K-carrying S_Alpha variant (S_Alpha+E484K) on lentivirus particles. (D) FRET histogram (left) and TDP (right) of ligand-free S_Alpha+E484K on lentivirus particles. (E and F) Experiments as in panels C and D, conducted in the presence of 200 μg/mL of hACE2. FRET histograms represent means ± SEM, determined from three randomly assigned populations of all FRET traces under corresponding experimental conditions. (G) Quantification of the FRET-indicated state occupancy for different spike variants. The occupancy in each FRET state was presented as mean ± SEM, determined by estimating the area under each Gaussian curve in FRET histograms. For fitting parameters, see Table S1.

FIG 4

D614G and E484K substitutions render S on the virus in favor of RBD-up open conformations, which are slower in temporal transitions. (A) Contour plots constructed from the compilation of the population of smFRET traces/trajectories. The contour plots were summed over time (15 s) of active/dynamic molecules compiled in the corresponding FRET histograms (Fig. 2C to F and Fig. 3A, B, D, and F). Some FRET-labeled active molecules that have reached photobleaching within 15 s contribute to the 0-FRET population. Contour plots blindly/unbiasedly monitor conformational ensembles occupied by virus-associated spike proteins over time. (B) Rates of transition between all observed FRET states for all tested S variants and their respective ligand-activated states. Rates are color-coded: warm color indicates relatively faster in transition, whereas cold color means slower in transition. The distribution of dwell times (Fig. S2 and S3) in each FRET state, determined through HMM, were fitted to the sum of two exponential distributions, y(t) = A1 exp^–k1t + A2 exp^–k2t, where y(t) is the probability and t is the dwell time. The weighted average of the two rate constants from each fit is presented. Error bars represent 95% confidence intervals propagated from the kinetics analysis.

FIG 5

Relative free-energy models depict conformational landscapes of different virus-associated spike variants upon activation by the binding of hACE2. (A to D) Free-energy models of parental S_D614 (A) and variants S_G614 (B), S_Alpha (C), and S_Alpha+E484K (D). The differences in free energies between states were roughly scaled based upon the relative state occupancies of each state. The ligand-free D614G- and E484K-carrying spikes are dominated by the intermediate-FRET state (one/two-RBD-up), which exhibits the lowest relative free energy among all four FRET states. In contrast, all hACE2-bound spikes show the lowest relative free energy at the low-FRET state (all-RBD-up).