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

Hsieh, C.-L.; Goldsmith, J. A.; Schaub, J. M.; DiVenere, A. M.; Kuo, H.-C.; Javanmardi, K.; Le, K. C.; Wrapp, D.; Lee, A. G.-W.; Liu, Y.; Chou, C.-W.; Byrne, P. O.; Hjorth, C. K.; Johnson, N. V.; Ludes-Meyers, J.; Nguyen, A. W.; Park, J.; Wang, N.; Dzifa, A.; Maynard, J. A.; Finkelstein, I. J.; McLellan, J. S.

doi:10.1101/2020.05.30.125484

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bioRxiv · 10.1101/2020.05.30.125484

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

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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 [A]-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.

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Hsieh, C.-L., Goldsmith, J. A., Schaub, J. M., DiVenere, A. M., Kuo, H.-C., Javanmardi, K., Le, K. C., Wrapp, D., Lee, A. G.-W., Liu, Y., Chou, C.-W., Byrne, P. O., Hjorth, C. K., Johnson, N. V., Ludes-Meyers, J., Nguyen, A. W., Park, J., Wang, N., Dzifa, A., Maynard, J. A., Finkelstein, I. J., McLellan, J. S.. 2020-05-30. Structure-based Design of Prefusion-stabilized SARS-CoV-2 Spikes. https://doi.org/10.1101/2020.05.30.125484

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