Structure-based design of prefusion-stabilized SARS-CoV-2 spikes
Ching-Lin Hsieh, Jory A Goldsmith, Jeffrey M Schaub, Andrea M DiVenere, Hung-Che Kuo, Kamyab Javanmardi, Kevin C Le, Daniel Wrapp, Alison G Lee, Yutong Liu, Chia-Wei Chou, Patrick O Byrne, Christy K Hjorth, Nicole V Johnson, John Ludes-Meyers, Annalee W Nguyen, Juyeon Park, Nianshuang Wang, Dzifa Amengor, Jason J Lavinder, Gregory C Ippolito, Jennifer A Maynard, Ilya J Finkelstein, Jason S McLellan, Ching-Lin Hsieh, Jory A Goldsmith, Jeffrey M Schaub, Andrea M DiVenere, Hung-Che Kuo, Kamyab Javanmardi, Kevin C Le, Daniel Wrapp, Alison G Lee, Yutong Liu, Chia-Wei Chou, Patrick O Byrne, Christy K Hjorth, Nicole V Johnson, John Ludes-Meyers, Annalee W Nguyen, Juyeon Park, Nianshuang Wang, Dzifa Amengor, Jason J Lavinder, Gregory C Ippolito, Jennifer A Maynard, Ilya J Finkelstein, Jason S McLellan
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).
Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.
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References
- Chan J. F. W., Yuan S., Kok K. H., To K. K. W., Chu H., Yang J., Xing F., Liu J., Yip C. C. Y., Poon R. W. S., Tsoi H. W., Lo S. K. F., Chan K. H., Poon V. K. M., Chan W. M., Ip J. D., Cai J. P., Cheng V. C. C., Chen H., Hui C. K. M., Yuen K. Y., A familial cluster of pneumonia associated with the 2019 novel coronavirus indicating person-to-person transmission: A study of a family cluster. Lancet 395, 514–523 (2020). 10.1016/S0140-6736(20)30154-9
- Huang C., Wang Y., Li X., Ren L., Zhao J., Hu Y., Zhang L., Fan G., Xu J., Gu X., Cheng Z., Yu T., Xia J., Wei Y., Wu W., Xie X., Yin W., Li H., Liu M., Xiao Y., Gao H., Guo L., Xie J., Wang G., Jiang R., Gao Z., Jin Q., Wang J., Cao B., Clinical features of patients infected with 2019 novel coronavirus in Wuhan, China. Lancet 395, 497–506 (2020). 10.1016/S0140-6736(20)30183-5
- Li F., Structure, Function, and Evolution of Coronavirus Spike Proteins. Annu. Rev. Virol. 3, 237–261 (2016). 10.1146/annurev-virology-110615-042301
- Hoffmann M., Kleine-Weber H., Schroeder S., Krüger N., Herrler T., Erichsen S., Schiergens T. S., Herrler G., Wu N. H., Nitsche A., Müller M. A., Drosten C., Pöhlmann S., SARS-CoV-2 Cell Entry Depends on ACE2 and TMPRSS2 and Is Blocked by a Clinically Proven Protease Inhibitor. Cell 181, 271–280.e8 (2020). 10.1016/j.cell.2020.02.052
- Wan Y., Shang J., Graham R., Baric R. S., Li F., Receptor Recognition by the Novel Coronavirus from Wuhan: An Analysis Based on Decade-Long Structural Studies of SARS Coronavirus. J. Virol. 94, e00127 (2020). 10.1128/JVI.00127-20
- Zhou P., Yang X. L., Wang X. G., Hu B., Zhang L., Zhang W., Si H. R., Zhu Y., Li B., Huang C. L., Chen H. D., Chen J., Luo Y., Guo H., Jiang R. D., Liu M. Q., Chen Y., Shen X. R., Wang X., Zheng X. S., Zhao K., Chen Q. J., Deng F., Liu L. L., Yan B., Zhan F. X., Wang Y. Y., Xiao G. F., Shi Z. L., A pneumonia outbreak associated with a new coronavirus of probable bat origin. Nature 579, 270–273 (2020). 10.1038/s41586-020-2012-7
- Bosch B. J., van der Zee R., de Haan C. A., Rottier J. M., The coronavirus spike protein is a class I virus fusion protein: Structural and functional characterization of the fusion core complex. J. Virol. 77, 8801–8811 (2003). 10.1128/JVI.77.16.8801-8811.2003
- Walls A. C., Tortorici M. A., Snijder J., Xiong X., Bosch B. J., Rey F. A., Veesler D., Tectonic conformational changes of a coronavirus spike glycoprotein promote membrane fusion. Proc. Natl. Acad. Sci. U.S.A. 114, 11157–11162 (2017). 10.1073/pnas.1708727114
- Buchholz U. J., Bukreyev A., Yang L., Lamirande E. W., Murphy B. R., Subbarao K., Collins P. L., Contributions of the structural proteins of severe acute respiratory syndrome coronavirus to protective immunity. Proc. Natl. Acad. Sci. U.S.A. 101, 9804–9809 (2004). 10.1073/pnas.0403492101
- Hofmann H., Hattermann K., Marzi A., Gramberg T., Geier M., Krumbiegel M., Kuate S., Überla K., Niedrig M., Pöhlmann S., S protein of severe acute respiratory syndrome-associated coronavirus mediates entry into hepatoma cell lines and is targeted by neutralizing antibodies in infected patients. J. Virol. 78, 6134–6142 (2004). 10.1128/JVI.78.12.6134-6142.2004
- Pallesen J., Wang N., Corbett K. S., Wrapp D., Kirchdoerfer R. N., Turner H. L., Cottrell C. A., Becker M. M., Wang L., Shi W., Kong W.-P., Andres E. L., Kettenbach A. N., Denison M. R., Chappell J. D., Graham B. S., Ward A. B., McLellan J. S., Immunogenicity and structures of a rationally designed prefusion MERS-CoV spike antigen. Proc. Natl. Acad. Sci. U.S.A. 114, E7348–E7357 (2017). 10.1073/pnas.1707304114
- Sanders R. W., Derking R., Cupo A., Julien J.-P., Yasmeen A., de Val N., Kim H. J., Blattner C., de la Peña A. T., Korzun J., Golabek M., de Los Reyes K., Ketas T. J., van Gils M. J., King C. R., Wilson I. A., Ward A. B., Klasse P. J., Moore J. P., A next-generation cleaved, soluble HIV-1 Env trimer, BG505 SOSIP.664 gp140, expresses multiple epitopes for broadly neutralizing but not non-neutralizing antibodies. PLOS Pathog. 9, e1003618 (2013). 10.1371/journal.ppat.1003618
- Crank M. C., Ruckwardt T. J., Chen M., Morabito K. M., Phung E., Costner P. J., Holman L. A., Hickman S. P., Berkowitz N. M., Gordon I. J., Yamshchikov G. V., Gaudinski M. R., Kumar A., Chang L. A., Moin S. M., Hill J. P., DiPiazza A. T., Schwartz R. M., Kueltzo L., Cooper J. W., Chen P., Stein J. A., Carlton K., Gall J. G., Nason M. C., Kwong P. D., Chen G. L., Mascola J. R., McLellan J. S., Ledgerwood J. E., Graham B. S., VRC 317 Study Team , A proof of concept for structure-based vaccine design targeting RSV in humans. Science 365, 505–509 (2019). 10.1126/science.aav9033
- Park Y. J., Walls A. C., Wang Z., Sauer M. M., Li W., Tortorici M. A., Bosch B. J., DiMaio F., Veesler D., Structures of MERS-CoV spike glycoprotein in complex with sialoside attachment receptors. Nat. Struct. Mol. Biol. 26, 1151–1157 (2019). 10.1038/s41594-019-0334-7
- Li Z., Tomlinson A. C. A., Wong A. H. M., Zhou D., Desforges M., Talbot P. J., Benlekbir S., Rubinstein J. L., Rini J. M., The human coronavirus HCoV-229E S-protein structure and receptor binding. eLife 8, e51230 (2019). 10.7554/eLife.51230
- Wang N., Rosen O., Wang L., Turner H. L., Stevens L. J., Corbett K. S., Bowman C. A., Pallesen J., Shi W., Zhang Y., Leung K., Kirchdoerfer R. N., Becker M. M., Denison M. R., Chappell J. D., Ward A. B., Graham B. S., McLellan J. S., Structural Definition of a Neutralization-Sensitive Epitope on the MERS-CoV S1-NTD. Cell Rep. 28, 3395–3405.e6 (2019). 10.1016/j.celrep.2019.08.052
- Walls A. C., Xiong X., Park Y.-J., Tortorici M. A., Snijder J., Quispe J., Cameroni E., Gopal R., Dai M., Lanzavecchia A., Zambon M., Rey F. A., Corti D., Veesler D., Unexpected Receptor Functional Mimicry Elucidates Activation of Coronavirus Fusion. Cell 176, 1026–1039.e15 (2019). 10.1016/j.cell.2018.12.028
- Wrapp D., Wang N., Corbett K. S., Goldsmith J. A., Hsieh C. L., Abiona O., Graham B. S., McLellan J. S., Cryo-EM structure of the 2019-nCoV spike in the prefusion conformation. Science 367, 1260–1263 (2020). 10.1126/science.abb2507
- Walls A. C., Park Y. J., Tortorici M. A., Wall A., McGuire A. T., Veesler D., Structure, Function, and Antigenicity of the SARS-CoV-2 Spike Glycoprotein. Cell 181, 281–292.e6 (2020). 10.1016/j.cell.2020.02.058
- McLellan J. S., Chen M., Joyce M. G., Sastry M., Stewart-Jones G. B. E., Yang Y., Zhang B., Chen L., Srivatsan S., Zheng A., Zhou T., Graepel K. W., Kumar A., Moin S., Boyington J. C., Chuang G.-Y., Soto C., Baxa U., Bakker A. Q., Spits H., Beaumont T., Zheng Z., Xia N., Ko S.-Y., Todd J.-P., Rao S., Graham B. S., Kwong P. D., Structure-based design of a fusion glycoprotein vaccine for respiratory syncytial virus. Science 342, 592–598 (2013). 10.1126/science.1243283
- Rutten L., Lai Y.-T., Blokland S., Truan D., Bisschop I. J. M., Strokappe N. M., Koornneef A., van Manen D., Chuang G.-Y., Farney S. K., Schuitemaker H., Kwong P. D., Langedijk J. P. M., A Universal Approach to Optimize the Folding and Stability of Prefusion-Closed HIV-1 Envelope Trimers. Cell Rep. 23, 584–595 (2018). 10.1016/j.celrep.2018.03.061
- Krarup A., Truan D., Furmanova-Hollenstein P., Bogaert L., Bouchier P., Bisschop I. J. M., Widjojoatmodjo M. N., Zahn R., Schuitemaker H., McLellan J. S., Langedijk J. P. M., A highly stable prefusion RSV F vaccine derived from structural analysis of the fusion mechanism. Nat. Commun. 6, 8143 (2015). 10.1038/ncomms9143
- Rutten L., Gilman M. S. A., Blokland S., Juraszek J., McLellan J. S., Langedijk J. P. M., Structure-Based Design of Prefusion-Stabilized Filovirus Glycoprotein Trimers. Cell Rep. 30, 4540–4550.e3 (2020). 10.1016/j.celrep.2020.03.025
- Battles M. B., Más V., Olmedillas E., Cano O., Vázquez M., Rodríguez L., Melero J. A., McLellan J. S., Structure and immunogenicity of pre-fusion-stabilized human metapneumovirus F glycoprotein. Nat. Commun. 8, 1528 (2017). 10.1038/s41467-017-01708-9
- Qiao H., Pelletier S. L., Hoffman L., Hacker J., Armstrong R. T., White J. M., Specific single or double proline substitutions in the “spring-loaded” coiled-coil region of the influenza hemagglutinin impair or abolish membrane fusion activity. J. Cell Biol. 141, 1335–1347 (1998). 10.1083/jcb.141.6.1335
- Hastie K. M., Zandonatti M. A., Kleinfelter L. M., Heinrich M. L., Rowland M. M., Chandran K., Branco L. M., Robinson J. E., Garry R. F., Saphire E. O., Structural basis for antibody-mediated neutralization of Lassa virus. Science 356, 923–928 (2017). 10.1126/science.aam7260
- Yuan M., Wu N. C., Zhu X., Lee C. D., So R. T. Y., Lv H., Mok C. K. P., Wilson I. A., A highly conserved cryptic epitope in the receptor binding domains of SARS-CoV-2 and SARS-CoV. Science 368, 630–633 (2020). 10.1126/science.abb7269
- Tegunov D., Cramer P., Real-time cryo-electron microscopy data preprocessing with Warp. Nat. Methods 16, 1146–1152 (2019). 10.1038/s41592-019-0580-y
- Punjani A., Rubinstein J. L., Fleet D. J., Brubaker M. A., cryoSPARC: Algorithms for rapid unsupervised cryo-EM structure determination. Nat. Methods 14, 290–296 (2017). 10.1038/nmeth.4169
- Ramírez-Aportela E., Vilas J. L., Glukhova A., Melero R., Conesa P., Martínez M., Maluenda D., Mota J., Jiménez A., Vargas J., Marabini R., Sexton P. M., Carazo J. M., Sorzano C. O. S., Automatic local resolution-based sharpening of cryo-EM maps. Bioinformatics 36, 765–772 (2020).
- Emsley P., Lohkamp B., Scott W. G., Cowtan K., Features and development of Coot. Acta Crystallogr. D 66, 486–501 (2010). 10.1107/S0907444910007493
- Liebschner D., Afonine P. V., Baker M. L., Bunkóczi G., Chen V. B., Croll T. I., Hintze B., Hung L. W., Jain S., McCoy A. J., Moriarty N. W., Oeffner R. D., Poon B. K., Prisant M. G., Read R. J., Richardson J. S., Richardson D. C., Sammito M. D., Sobolev O. V., Stockwell D. H., Terwilliger T. C., Urzhumtsev A. G., Videau L. L., Williams C. J., Adams P. D., Macromolecular structure determination using X-rays, neutrons and electrons: Recent developments in Phenix. Acta Crystallogr. D 75, 861–877 (2019). 10.1107/S2059798319011471
- Croll T. I., ISOLDE: A physically realistic environment for model building into low-resolution electron-density maps. Acta Crystallogr. D 74, 519–530 (2018). 10.1107/S2059798318002425
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