Structure-based Design of Prefusion-stabilized SARS-CoV-2 Spikes

Abstract

The COVID-19 pandemic caused by the novel coronavirus SARS-CoV-2 has led to accelerated efforts to develop therapeutics, diagnostics, and vaccines to mitigate this public health emergency. A key target of these efforts is the spike (S) protein, a large trimeric class I fusion protein that is metastable and difficult to produce recombinantly in large quantities. Here, we designed and expressed over 100 structure-guided spike variants based upon a previously determined cryo-EM structure of the prefusion SARS-CoV-2 spike. Biochemical, biophysical and structural characterization of these variants identified numerous individual substitutions that increased protein yields and stability. The best variant, HexaPro, has six beneficial proline substitutions leading to ~10-fold higher expression than its parental construct and is able to withstand heat stress, storage at room temperature, and multiple freeze-thaws. A 3.2 Å-resolution cryo-EM structure of HexaPro 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 SARS-CoV-2.

Figure 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).

Figure 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-D) Size exclusion chromatography 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) Concentrations of individual variants in culture medium, 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.

Figure 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, the green vertical dotted line shows the first apparent melting temperature for Combo47 (HexaPro). (D) Negative stain electron micrograph of purified Combo47 (HexaPro).

Figure 4.. HexaPro exhibits enhanced expression and stability compared to S-2P.

(A) SEC trace of a portion of the HexaPro purified from a 2L culture of FreeStyle 293-F cells. (B) Negative stain electron micrograph of HexaPro purified from FreeStyle 293-F cells. (C) SEC trace of HexaPro after purification from a 40 ml culture of ExpiCHO cells. (D) Negative stain electron micrograph of HexaPro from ExpiCHO cells. (E-F) Binding of S-2P (E) and HexaPro (F) to human ACE2 assessed by surface plasmon resonance. Binding data are shown as black lines and the best fit to a 1:1 binding model is shown as red lines. (G-H) Assessment of protein stability by negative stain electron microscopy. The top row of micrographs in (G) and (H) corresponds to S-2P, the bottom row corresponds to HexaPro.

Figure 5.. 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, individual atoms are shown as sticks. Nitrogen atoms are colored blue and oxygen atoms are colored red.