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

Area of Science:

• Virology
• Structural Biology
• Protein Engineering

Background:

• The COVID-19 pandemic necessitates rapid development of vaccines and therapeutics.
• The SARS-CoV-2 spike (S) protein is a critical target but challenging to produce recombinantly due to its metastability.
• Previous cryo-electron microscopy (cryo-EM) structures provide a basis for protein engineering.

Approach:

• Over 100 structure-guided spike protein variants were designed and expressed using cryo-electron microscopy (cryo-EM) data.
• Biochemical, biophysical, and structural characterization methods were employed to assess variant stability and yield.
• Site-directed mutagenesis was used to introduce stabilizing proline substitutions.

Key Points:

• HexaPro, a variant with six proline substitutions, demonstrated approximately 10-fold higher expression levels compared to the parental construct.
• HexaPro exhibits enhanced stability, tolerating heat stress, room temperature storage, and multiple freeze-thaw cycles.
• A 3.2 Å cryo-EM structure confirmed that HexaPro maintains the desired prefusion spike conformation.

Conclusions:

• High-yield production of a stabilized prefusion SARS-CoV-2 spike protein is achievable.
• The HexaPro variant offers a robust platform for vaccine and diagnostic development.
• This work accelerates the response to the COVID-19 pandemic through improved biological reagents.