Enhancing the Prefusion Conformational Stability of SARS-CoV-2 Spike Protein Through Structure-Guided Design

Abstract

The worldwide pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is unprecedented and the impact on public health and the global economy continues to be devastating. Although early therapies such as prophylactic antibodies and vaccines show great promise, there are concerns about the long-term efficacy and universal applicability of these therapies as the virus continues to mutate. Thus, protein-based immunogens that can quickly respond to viral changes remain of continued interest. The Spike protein, the main immunogen of this virus, displays a highly dynamic trimeric structure that presents a challenge for therapeutic development. Here, guided by the structure of the Spike trimer, we rationally design new Spike constructs that show a uniquely high stability profile while simultaneously remaining locked into the immunogen-desirable prefusion state. Furthermore, our approach emphasizes the relationship between the highly conserved S2 region and structurally dynamic Receptor Binding Domains (RBD) to enable vaccine development as well as the generation of antibodies able to resist viral mutation.

Keywords: COVID-19; SARS-CoV-2; prefusion; spike; trimer stability.

Figure 1

Deconstruction of the SARS-CoV-2 Spike Trimer. Flowchart representation of the experimental variables in this study consisting of (top to bottom): trimerization domain, furin site, K986P/V987P prefusion locking mutations, and rationally designed disulfides (A). Structural models of each rationally designed disulfide, with sequence maps shown below each model (B). In (B), engineered disulfides are shown as spheres in each structural model, and the disulfide-linked regions are indicated in each sequence map by a yellow band.

Figure 2

Identification of spike trimers. Expanded view of the Spike trimer, highlighting the fibritin trimerization domain, furin cleavage site and abrogation mutations, and the prefusion stabilizing mutations K986P/V987P (A). The effects of stabilization mutations (Spike_PP), removal of the trimerization domain (Spike_Monomer) and re-introduction of the furin cleavage site (Spike_Furin) on the oligomerization state of the Spike protein compared to Spike_KV (Superose 6 SEC chromatograms; Standard elution traces shown in grey) (B). Negative stain EM images of Spike_Monomer (top), Spike_KV (middle) and Spike_PP (bottom) showing different trimerization and stabilization properties, with zoom-in of individual particles outlined in red; (C). 2D class average (above) and 3D reconstruction (below) of the Spike_KV construct reveal two distinct Spike trimer populations, closed and partial open (D). Negative stain EM images of Spike_KV beyond Day 5 after purification show significant particle degradation (E). Scale bars in (C) and (E) indicate 100nm, and white circles highlight individual particles.

Figure 3

Trimer formation of rationally designed disulfides. SEC chromatograms (left) and corresponding structural models (right, with zoom-in) of each of the top four disulfide designs. RCC3 (A), RCC4 (B), RCC5 (C), and RCC6 (D) each migrate on the SEC primarily as a trimer with a peak corresponding to a 669kDa MW standard (Standard elution traces shown in grey). Residues that form the engineered disulfide are shown as spheres the overall view of each model, and as sticks in each zoomed-in view.

Figure 4

ACE2 Binding and pseudovirus inhibition. Expression of Spike trimer on cell surface of HEK293 cells as determined by staining with anti-S protein antibody (A). Normalized ACE2 binding of cells expressing Spike constructs as determined by staining with ACE2-Fc antigen (B). Luciferase activity of HEK293 cells infected with pseudovirus particles displaying Spike constructs with a Luciferase reporter gene. An unpaired t-test indicates the Spike_PP activity is significantly inhibited compared to Spike_KV. Additionally, p-values indicate a significant difference between Spike_PP and RCC3 as well (C). Model of Spike trimer postfusion transition highlight two possible stages [(1) and (2)] for postfusion inhibition (modeled from PDB 6VSB and 6M3W) (6, 36) (D). All experiments performed in triplicate with error bars denoting the standard deviation. Note: RCC3_KV design failed cloning assembly. ***p<=0.001.

Figure 5

RCC3 locks All RBDs into a singular orientation. Schematic representation of RCC3 disulfide mutation expected RBD behavior (modeled from PDB 6VXX), viewed from the “top” (left) and “side” (center) and zoomed in to show the engineered disulfide (right) (A). 2D class averages (top) and 3D reconstruction (bottom) of negative stain EM images of the RCC3_KV (B) and RCC3_PP (C) designs demonstrate all particles contained all three RBDs in the “closed” configuration without disrupting the S2 subunit. Spike particles were observed by negative stain EM images to be stable without significant degradation up to 27 days (D). Scale bars in (D) indicate 100nm and white circles highlight individual particles.

Figure 6

RCC6 stabilizes the prefusion state independent of RBD orientation. Schematic representation of RCC6 disulfide mutation and expected RBD behavior (modeled from PDB 6VXX and 6VSB) viewed from the “top” (left) and “side” (center) and zoomed in to show the engineered disulfide (right) (A). 2D class averages (top) and 3D reconstruction (bottom) of negative stain EM images of the RCC6_KV designs shows ~45% of the particles maintain all RBDs in the “closed” configuration, while ~55% of particles are partially “open” (B). In contrast, the RCC6_PP design resulted in 47% of particles in the partially “open” state and 53% in a unique “full open” state with no RBDs visible (C). Both RCC6_KV and RCC6_PP had no apparent effect on S2 organization. Similar to RCC3, particles containing the RCC6 design could be identified by negative stain EM images up to 27 days with no apparent degradation (D). Scale bars in (D) indicate 100nm and white circles highlight individual particles.