A thermostable, closed SARS-CoV-2 spike protein trimer

The spike (S) protein of SARS-CoV-2 mediates receptor binding and cell entry and is the dominant target of the immune system. S exhibits substantial conformational flexibility. It transitions from closed to open conformations to expose its receptor binding site, and subsequently from prefusion to postfusion conformations to mediate fusion of viral and cellular membranes. S protein derivatives are components of vaccine candidates and diagnostic assays, as well as tools for research into the biology and immunology of SARS-CoV-2. Here we have designed mutations in S which allow production of thermostable, crosslinked, S protein trimers that are trapped in the closed, pre-fusion, state. We have determined the structures of crosslinked and non-crosslinked proteins, identifying two distinct closed conformations of the S trimer. We demonstrate that the designed, thermostable, closed S trimer can be used in serological assays. This protein has potential applications as a reagent for serology, virology and as an immunogen.

John A.G. Briggs | Ian Goodfellow | Andrew P. Carter | Xiaoli Xiong | Ying Zhang | Myra Hosmillo | Kenneth G. C. Smith | James A. Nathan | Rainer Doffinger | S. Baker | J. Briggs | X. Xiong | Nigel R. Ovington | S. Scheres | L. Pointon | P. Lyons | L. James | A. Carter | S. Graf | Jennifer M. Martin | I. Goodfellow | L. Bergamaschi | K. Qu | Zunlong Ke | B. Dunmore | H. Baxendale | S. Papadia | R. Doffinger | H. Butcher | C. Treacy | M. Toshner | F. Mescia | H. Stark | Stephen Baker | E. Jones | K. Hunter | Laura Bergamaschi | M. Epping | E. Legchenko | Sjors H.W. Scheres | Leo C. James | K. Stirrups | Zunlong Ke | Kun Qu | Ciara O’Donnell | A. Elmer | Helen E. Baxendale | S. Hewitt | Katarzyna A. Ciazynska | Soraya Ebrahimi | Guinevere L. Grice | J. Nathan | M. Hosmillo | J. Bradley | G. Grice | Marta Wylot | J. Rowlands | N. Richoz | Nicole Pond | Rebecca J Rastall | Sherly Jose | Jennifer M Martin | Neil E. Walker | Emily Li | J. Kennet | Aloka De Sa | B. Graves | J. Shih | O. Huhn | Stuart Fawke | Isobel Jarvis | S. Ebrahimi | M. Perera | Ashlea Bucke | J. Calder | L. Canna | Julie Harris | Jenny Kourampa | A. Meadows | Criona O’Brien | Jane Price | C. Ribeiro | Valentina Ruffolo | Ben Bullman | Christopher Huang | R. Sutcliffe | Z. Tong | Ariana Betancourt | G. Bower | F. Nice | Ommar Omarjee | Ying Zhang | T. Tilly | K. Ciazynska | Cherry Crucusio | Hugo Tordesillas | Emma Le Gresley | J. Marsden | Rahul Sharma | Eleanor Dewhust | Jason Domingo | L. Turner | D. Caputo | Josh Hodgson | Jennifer Wood | John Paul A. Kenneth G. C. Mark Anne Carla Jenny Sherly Bradley Lyons Smith Toshner Elmer Ribeiro Ko | Marianne R Perera | B. Bullman | Francesca L. Nice | J. Bradley

[1]  Dariusz M Plewczynski,et al.  Three-dimensional Epigenome Statistical Model: Genome-wide Chromatin Looping Prediction , 2018, Scientific Reports.

[2]  Daniel Wrapp,et al.  Stabilized coronavirus spikes are resistant to conformational changes induced by receptor recognition or proteolysis , 2018, Scientific Reports.

[3]  Frank DiMaio,et al.  Glycan shield and epitope masking of a coronavirus spike protein observed by cryo-electron microscopy , 2016, Nature Structural &Molecular Biology.

[4]  P. Kwong,et al.  How HIV-1 entry mechanism and broadly neutralizing antibodies guide structure-based vaccine design , 2017, Current opinion in HIV and AIDS.

[5]  David N Mastronarde,et al.  Automated electron microscope tomography using robust prediction of specimen movements. , 2005, Journal of structural biology.

[6]  Haixia Zhou,et al.  Cryo-electron microscopy structures of the SARS-CoV spike glycoprotein reveal a prerequisite conformational state for receptor binding , 2016, Cell Research.

[7]  A. Walls,et al.  Closing coronavirus spike glycoproteins by structure-guided design , 2020, bioRxiv.

[8]  Alan A. Dombkowski,et al.  Disulfide by Design 2.0: a web-based tool for disulfide engineering in proteins , 2013, BMC Bioinformatics.

[9]  Linqi Zhang,et al.  Human neutralizing antibodies elicited by SARS-CoV-2 infection , 2020, Nature.

[10]  Sjors H W Scheres,et al.  Estimation of high-order aberrations and anisotropic magnification from cryo-EM data sets in RELION-3.1 , 2020, IUCrJ.

[11]  A. Walls,et al.  Tectonic conformational changes of a coronavirus spike glycoprotein promote membrane fusion , 2017, Proceedings of the National Academy of Sciences.

[12]  Kaijun Jiang,et al.  SARS‐CoV‐2 Seroconversion in Humans: A Detailed Protocol for a Serological Assay, Antigen Production, and Test Setup , 2020, Current protocols in microbiology.

[13]  Shaun Rawson,et al.  Distinct conformational states of SARS-CoV-2 spike protein , 2020, Science.

[14]  D. Agard,et al.  MotionCor2: anisotropic correction of beam-induced motion for improved cryo-electron microscopy , 2017, Nature Methods.

[15]  Poul Nissen,et al.  Namdinator – automatic molecular dynamics flexible fitting of structural models into cryo-EM and crystallography experimental maps , 2019, IUCrJ.

[16]  Poul Nissen,et al.  Namdinator – automatic molecular dynamics flexible fitting of structural models into cryo-EM and crystallography experimental maps , 2018, bioRxiv.

[17]  Yi Shi,et al.  Cryo-EM structures of MERS-CoV and SARS-CoV spike glycoproteins reveal the dynamic receptor binding domains , 2017, Nature Communications.

[18]  Jason S McLellan,et al.  Structure-Based Vaccine Antigen Design. , 2019, Annual review of medicine.

[19]  Jing Yuan,et al.  Viral Architecture of SARS-CoV-2 with Post-Fusion Spike Revealed by Cryo-EM , 2020, bioRxiv.

[20]  M. Borgnia,et al.  Controlling the SARS-CoV-2 Spike Glycoprotein Conformation , 2020, bioRxiv.

[21]  Barney S. Graham,et al.  Immunogenicity and structures of a rationally designed prefusion MERS-CoV spike antigen , 2017, Proceedings of the National Academy of Sciences.

[22]  Thibaut Jombart,et al.  When are pathogen genome sequences informative of transmission events? , 2018, PLoS pathogens.

[23]  A. Walls,et al.  Unexpected Receptor Functional Mimicry Elucidates Activation of Coronavirus Fusion , 2019, Cell.

[24]  A. Bhardwaj,et al.  In situ click chemistry generation of cyclooxygenase-2 inhibitors , 2017, Nature Communications.

[25]  David I Stuart,et al.  Neutralization of SARS-CoV-2 by Destruction of the Prefusion Spike , 2020, bioRxiv.

[26]  John P. Moore,et al.  A Next-Generation Cleaved, Soluble HIV-1 Env Trimer, BG505 SOSIP.664 gp140, Expresses Multiple Epitopes for Broadly Neutralizing but Not Non-Neutralizing Antibodies , 2013, PLoS pathogens.

[27]  William J. Liu,et al.  A human neutralizing antibody targets the receptor-binding site of SARS-CoV-2 , 2020, Nature.

[28]  Fang Li,et al.  Cryo-EM structure of infectious bronchitis coronavirus spike protein reveals structural and functional evolution of coronavirus spike proteins , 2018, PLoS pathogens.

[29]  C. Cunningham-Rundles,et al.  A serological assay to detect SARS-CoV-2 seroconversion in humans , 2020, Nature Medicine.

[30]  A. Walls,et al.  Structure, Function, and Antigenicity of the SARS-CoV-2 Spike Glycoprotein , 2020, Cell.

[31]  Christian Drosten,et al.  Severe acute respiratory syndrome-related coronavirus: The species and its viruses – a statement of the Coronavirus Study Group , 2020, bioRxiv.

[32]  D. Montefiori,et al.  Spike mutation pipeline reveals the emergence of a more transmissible form of SARS-CoV-2 , 2020, bioRxiv.

[33]  Randy J Read,et al.  Real-space refinement in PHENIX for cryo-EM and crystallography , 2018, bioRxiv.

[34]  Xinquan Wang,et al.  Cryo-EM structure of the SARS coronavirus spike glycoprotein in complex with its host cell receptor ACE2 , 2018, PLoS pathogens.

[35]  N. Grigorieff,et al.  CTFFIND4: Fast and accurate defocus estimation from electron micrographs , 2015, bioRxiv.

[36]  B. Graham,et al.  Cryo-EM structure of the 2019-nCoV spike in the prefusion conformation , 2020, Science.

[37]  I. Wilson,et al.  A highly conserved cryptic epitope in the receptor binding domains of SARS-CoV-2 and SARS-CoV , 2020, Science.

[38]  Yang Yang,et al.  A noncompeting pair of human neutralizing antibodies block COVID-19 virus binding to its receptor ACE2 , 2020, Science.

[39]  F. Dimaio,et al.  Cryo-electron microscopy structure of a coronavirus spike glycoprotein trimer , 2016, Nature.

[40]  Cinque S. Soto,et al.  Structure-Based Design of a Fusion Glycoprotein Vaccine for Respiratory Syncytial Virus , 2013, Science.

[41]  Kevin Cowtan,et al.  research papers Acta Crystallographica Section D Biological , 2005 .

[42]  Fang Li,et al.  Molecular Mechanism for Antibody-Dependent Enhancement of Coronavirus Entry , 2019, Journal of Virology.

[43]  Kai Zhao,et al.  A pneumonia outbreak associated with a new coronavirus of probable bat origin , 2020, Nature.

[44]  Qiang Zhou,et al.  A potent neutralizing human antibody reveals the N-terminal domain of the Spike protein of SARS-CoV-2 as a site of vulnerability , 2020, bioRxiv.

[45]  Glycan Shield and Fusion Activation of a Deltacoronavirus Spike Glycoprotein Fine-Tuned for Enteric Infections , 2017, Journal of Virology.

[46]  G. Gao,et al.  A Novel Coronavirus from Patients with Pneumonia in China, 2019 , 2020, The New England journal of medicine.

[47]  Jasenko Zivanov,et al.  A Bayesian approach to beam-induced motion correction in cryo-EM single-particle analysis , 2018, bioRxiv.

[48]  Fang Li,et al.  Structure, Function, and Evolution of Coronavirus Spike Proteins. , 2016, Annual review of virology.