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

Abstract

SARS-CoV-2 spike (S) mediates entry into cells and is critical for vaccine development against COVID-19. S is synthesized as a precursor, processed into S1 and S2 by furin proteases, and activated for fusion when human angiotensin-converting enzyme 2 (hACE2) engages the receptor-binding domain (RBD) and when the N-terminus of S2 is proteolytically processed. Structures of soluble ectodomains and native virus particles have revealed distinct conformations of S, including a closed trimer with all RBD oriented downward, trimers with one or two RBDs up, and hACE2-stabilized conformations with up to three RBD oriented up. Real-time information that connects these structures, however, has been lacking. Here we apply single-molecule Forster 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 hACE2, S opens into the hACE2-bound S conformation through at least one on-path intermediate, with trypsin partially activating S. Conformational preferences of convalescent patient plasma and monoclonal antibodies suggest mechanisms of neutralization involving either direct competition with hACE2 for binding to RBD or allosteric interference with conformational changes required for entry. Our findings inform on mechanisms of S recognition and on conformations for immunogen design.

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

“…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.…”

FRETing over SARS-CoV-2: Conformational Dynamics of the Spike Glycoprotein

“…In this issue of Cell Host & Microbe , Lu et al. (2020) used smFRET to study the in situ conformational dynamics of the SARS-CoV-2 S glycoprotein on the surface of lentivirus pseudotyped and coronavirus-like particles.…”

Coevolutionary Analysis and Perturbation-Based Network Modeling of the SARS-CoV-2 Spike Protein Complexes with Antibodies: Binding-Induced Control of Dynamics, Allosteric Interactions and Signaling

“…Biophysical analysis of SARS-CoV-2 S trimer on virus particles revealed four distinct conformational states for the S protein and a sequence of conformational transitions through an obligatory intermediate in which all three RBD domains in the closed conformations are oriented towards the viral particle membrane. 33 Cryo-EM structural studies also mapped a mechanism of conformational events associated with ACE2 binding, showing that the compact closed form of the SARS-CoV-2 S protein becomes weakened after furin cleavage between the S1 and S2…”

“…Biophysical analysis of SARS-CoV-2 S trimer on virus particles revealed four distinct conformational states for the S protein and a sequence of conformational transitions through an obligatory intermediate in which all three RBD domains in the closed conformations are oriented towards the viral particle membrane. 33 Cryo-EM structural studies also mapped a mechanism of conformational events associated with ACE2 binding, showing that the compact closed form of the SARS-CoV-2 S protein becomes weakened after furin cleavage between the S1 and S2 domains, leading to the increased population of partially open states and followed by ACE2 recognition that can accelerate transformation to a fully open and ACE2-bound form priming the protein for fusion activation. 34 The early biochemical studies of SARS S proteins with antibodies (Abs) suggested that RBD regions of S proteins contain multiple conformation-dependent epitopes capable of inducing potent neutralizing Ab responses, thus revealing the link between conformational heterogeneity of S proteins and capacity for eliciting binding with highly potent neutralizing Abs.…”