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

Hsieh, Ching-Lin; Goldsmith, Jory A.; Schaub, Jeffrey M.; DiVenere, Andrea M.; Kuo, Hung-Che; Javanmardi, Kamyab; Le, Kevin C.; Wrapp, Daniel; Lee, Alison Gene-Wei; Liu, Yutong; Chou, Chia-Wei; Byrne, Patrick O.; Hjorth, Christy K.; Johnson, Nicole V.; Ludes-Meyers, John; Nguyen, Annalee W.; Park, Juyeon; Wang, Nianshuang; Amengor, Dzifa; Maynard, Jennifer A.; Finkelstein, Ilya J.; McLellan, Jason S.

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 Ã…-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.