Real-Time Conformational Dynamics of SARS-CoV-2 Spikes on Virus Particles

Abstract

SARS-CoV-2 spike (S) mediates viral entry into cells and is critical for vaccine development against COVID-19. Structural studies have revealed distinct conformations of S, but real-time information that connects these structures, is lacking. Here we apply single-molecule Fluorescence (Förster) Resonance Energy Transfer (smFRET) imaging to observe conformational dynamics of S on virus particles. Virus-associated S dynamically samples at least four distinct conformational states. In response to human receptor Angiotensin-Converting Enzyme 2 (hACE2), S opens sequentially into the hACE2-bound S conformation through at least one on-path intermediate. Conformational preferences observed upon expsoure to convalescent plasma or antibodies suggest mechanisms of neutralization involving either competition with hACE2 for binding to the receptor-binding domain (RBD) or allosteric interference with conformational changes required for entry. Our findings inform on mechanisms of S recognition and conformations for immunogen design.

A glycan gate controls opening of the SARS-CoV-2 spike protein

“…4,5,6 The RBDs must transition from a down to an up state for the receptor binding motif to be accessible for ACE2 binding (Figure 1), and therefore the activation mechanism is essential for cell entry. Mothes et al 7 used single-molecule fluorescence (Förster) resonance energy transfer (smFRET) imaging to characterize spike dynamics in real-time. Their work showed that the spike dynamically visits four distinct conformational states, the population of which are modulated by the presence of the human ACE2 receptor and antibodies.…”

Kobophenol A Inhibits Binding of Host ACE2 Receptor with Spike RBD Domain of SARS-CoV-2, a Lead Compound for Blocking COVID-19

“…This study shows the movement of activation spike protein from the ground state to the activated state via an intermediate by hACE2 and suggested proteolytic processing of spike accelerates hACE2 dependent activation using single-molecule fluorescence resonance energy transfer (smFRET) imaging. 8 Interestingly, a highly flexible receptor-binding domain (RBD) of the S-protein was identified to be locked in either “down” or “up” state conformations. 9 These findings suggest the importance of conformation control via rational design for spike protein and can be applied to engineer vaccine against SARS-CoV-2 S proteins.…”

Single-Molecule FRET Imaging of Virus Spike–Host Interactions

“…The genetically encoded copper-free click chemistry (amber-click) allows reading through introduced amber stop codons on the protein of interest as unnatural amino acids through amber suppression, followed by site-specifically labeling conjugated dyes on unnatural amino acids by copper-free click chemistry [ 55 , 56 ]. Both enzymatic and amber-click methods have been applied to study virus spikes for many enveloped viruses to reveal dynamic aspects during viral membrane fusion [ 22 , 23 , 24 , 25 , 26 , 27 , 28 , 29 , 30 , 31 ].…”

“…Numerous pre-fusion and post-fusion structures of virus spikes have provided unprecedented details of conformations at individual steps during the viral membrane fusion [ 5 , 6 , 7 , 8 , 9 , 10 , 11 , 12 , 13 , 14 , 15 , 16 , 17 , 18 , 19 , 20 , 21 ]. The recent dynamic studies on virus spikes established platforms to connect these structural snapshots in real time, revealed the order and the kinetics of transitional events, and guided developing interventions aiming to arrest or block viral membrane fusion, thus stopping viral infection [ 22 , 23 , 24 , 25 , 26 , 27 , 28 , 29 , 30 , 31 ].…”

“…Single-molecule Förster resonance energy transfer (smFRET), a spectroscopic tool sensitive to inter-fluorophore molecular distances, has been well-positioned to capture the innate dynamics of virus spikes labeled with Förster resonance energy transfer (FRET)-paired fluorophores. This minireview covers recent work on single-molecule FRET imaging of class I fusion proteins of enveloped viruses, including SARS-CoV-2 [ 31 ], HIV-1 [ 22 , 23 , 24 , 25 , 26 , 27 ], influenza [ 29 ], and Ebola [ 28 , 30 ].…”