Structure-based design of prefusion-stabilized SARS-CoV-2 spikes

Abstract

The coronavirus disease 2019 (COVID-19) pandemic has led to accelerated efforts to develop therapeutics and vaccines. A key target of these efforts is the spike (S) protein, which is metastable and difficult to produce recombinantly. We characterized 100 structure-guided spike designs and identified 26 individual substitutions that increased protein yields and stability. Testing combinations of beneficial substitutions resulted in the identification of HexaPro, a variant with six beneficial proline substitutions exhibiting higher expression than its parental construct (by a factor of 10) as well as the ability to withstand heat stress, storage at room temperature, and three freeze-thaw cycles. A cryo-electron microscopy structure of HexaPro at a resolution of 3.2 angstroms confirmed that it retains the prefusion spike conformation. High-yield production of a stabilized prefusion spike protein will accelerate the development of vaccines and serological diagnostics for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

Fig. 1. Exemplary substitutions for SARS-CoV-2 spike stabilization.

Side view of the trimeric SARS-CoV-2 spike ectodomain in a prefusion conformation (PDB ID: 6VSB). The S1 domains are shown as a transparent molecular surface. The S2 domain for each protomer is shown as a ribbon diagram. Each inset corresponds to one of four types of spike modifications (proline, salt bridge, disulfide, cavity filling). Side chains in each inset are shown as red spheres (proline), yellow sticks (disulfide), red and blue sticks (salt bridge) and orange spheres (cavity filling).

Fig. 2. Characterization of single-substitution spike variants.

(A) SDS-PAGE of SARS-CoV-2 S-2P and single-substitution spike variants. Molecular weight standards are indicated at the left in kDa. (B to D) Size-exclusion chromatography traces of purified spike variants, grouped by type (B, disulfide variants; C, cavity filling and salt bridge; D, proline). A vertical dotted line indicates the characteristic peak retention volume for S-2P. (E) Representative negative stain electron micrographs for four variants. (F) Differential scanning fluorimetry analysis of spike variant thermostability. The vertical dotted line indicates the first apparent melting temperature for S-2P. (G) Expression levels of individual variants determined by quantitative biolayer interferometry. Variants are colored by type. The horizontal dotted line indicates the calculated concentration of S-2P, which was used as a control for comparison. The mean of three biological replicates is plotted, with error bars indicating standard deviations.

Fig. 3

Characterization of multi-substitution spike variants. (A) SDS-PAGE of SARS-CoV-2 Combo variants. Molecular weight standards are indicated at the left in kDa. (B) SEC traces for S-2P, A892P and four Combo variants. The vertical dotted line indicates the peak retention volume for S-2P. (C) DSF analysis of Combo variant thermostability. The black vertical dotted line indicates the first apparent melting temperature for S-2P and the green vertical dotted line indicates the first apparent melting temperature for Combo47 (HexaPro). (D) Negative stain electron micrograph of purified Combo47 (HexaPro). (E) Binding of S-2P or HexaPro to convalescent human sera, mAb CR3022 and negative control serum (GNEG), measured by ELISA.

Fig. 4

High-resolution cryo-EM structure of HexaPro. (A) EM density map of trimeric HexaPro. Each protomer is shown in a different color; the protomer depicted in wheat adopts the RBD-up conformation. (B) Alignment of an RBD-down protomer from HexaPro (green ribbon) with an RBD-down protomer from S-2P (white ribbon, PDB ID: 6VSB). (C) Zoomed view of the four proline substitutions unique to HexaPro. The EM density map is shown as a transparent surface and individual atoms are shown as sticks. Nitrogen atoms are colored blue and oxygen atoms are colored red.