Engineered disulfide reveals structural dynamics of locked SARS-CoV-2 spike

Abstract

The spike (S) protein of SARS-CoV-2 has been observed in three distinct pre-fusion conformations: locked, closed and open. Of these, the function of the locked conformation remains poorly understood. Here we engineered a SARS-CoV-2 S protein construct “S-R/x3” to arrest SARS-CoV-2 spikes in the locked conformation by a disulfide bond. Using this construct we determined high-resolution structures confirming that the x3 disulfide bond has the ability to stabilize the otherwise transient locked conformations. Structural analyses reveal that wild-type SARS-CoV-2 spike can adopt two distinct locked-1 and locked-2 conformations. For the D614G spike, based on which all variants of concern were evolved, only the locked-2 conformation was observed. Analysis of the structures suggests that rigidified domain D in the locked conformations interacts with the hinge to domain C and thereby restrains RBD movement. Structural change in domain D correlates with spike conformational change. We propose that the locked-1 and locked-2 conformations of S are present in the acidic high-lipid cellular compartments during virus assembly and egress. In this model, release of the virion into the neutral pH extracellular space would favour transition to the closed or open conformations. The dynamics of this transition can be altered by mutations that modulate domain D structure, as is the case for the D614G mutation, leading to changes in viral fitness. The S-R/x3 construct provides a tool for the further structural and functional characterization of the locked conformations of S, as well as how sequence changes might alter S assembly and regulation of receptor binding domain dynamics.

SARS-CoV-2 Delta and Omicron variants evade population antibody response by mutations in a single spike epitope

“…S-R/PP (2P-stabilized spike with furin site changed to a single R) and S-R (unstabilized native spike with furin site changed to a single R) have been shown to exist primarily in 1 RBD ‘up’ (80% 1 RBD ‘up’, 20% closed) and unrestrained closed (20% 1 RBD ‘up’, 80% closed) conformations, respectively 8 . RBDs in S-R/x2 and S-R/x3 spikes were restrained by the x2 and x3 disulfide bonds in ‘down’ positions, and the two spikes differ slightly in trimer packing, adopting primarily closed 8 and locked 42 conformations, respectively. IgGs were immobilized onto Protein A biosensors and submerged into 2-fold serially diluted RBD and spike solutions (200, 100, 50, 25, 12.5, 6.25, 3.125 nM) to record sensorgrams (black lines).…”

“…S-GSAS/6P was constructed according to a previous report 41 ; all other SARS-CoV-2 S and ACE2-Fc constructs have been previously described and were expressed and purified following the previously established protocols 8 , 42 , 43 . To obtain ACE2 without tag (referred to as ACE2), the Fc tag was removed by trypsin digestion in a reaction mixture containing 5 mg ACE2-Fc and 50 µg trypsin in PBS.…”

“… 8 ) and x3 disulfide stabilized RBD ‘down’ (S-R/x3) spikes (ref. 42 ) were compared to the corresponding Delta variant spikes (S-R/Delta, S-R/x3/Delta). Kinetic parameters are summarized in Supplementary Table 7 .…”

Disulfide stabilization reveals conserved dynamic features between SARS-CoV-1 and SARS-CoV-2 spikes

“…Detailed structural analysis of SARS-CoV-2 S structures identified that interactions between Domain D and Domain C-D hinge region in locked conformations likely restrain RBD movement leading to more compact spike packing (Qu et al , 2022). A comparison to other CoV S structures revealed that HCoV-NL63 (Walls et al ., 2016b), MHV (Walls et al ., 2016a), Infectious Bronchitis Virus (IBV) (Shang et al , 2018) and Porcine Delta coronavirus (PDCoV) (Xiong et al , 2018) spikes are structurally more related to locked structures of SARS-CoV-2 spike, they all have ordered FPPR which is incompatible with RBD in “up” position (Xiong et al ., 2020).…”

“…The three-fold axes are indicated by black triangles. Locked SARS-CoV-1 spikes (a-d; f and h), animal SARSr-CoV spikes (i, EMD-12130(Wrobel et al, 2021); j, EMD-30418(Zhang et al, 2021b); k, EMD30416(Zhang et al, 2021b); l, EMD-11204(Wrobel et al, 2020)) and locked SARS-CoV-2 spikes (m, EMD-33453(Qu et al, 2022); n,…”