In brief, virus stock (9.75 × 104 p.f.u. Destabilization of viral proteins by antibodies has been observed for influenza19 and human immunodeficiency virus20. H11-D4 is colored orange and H11-H4 yellow. The rapid pipeline from naive library screen to maturation and thorough characterization does offer the possibility that new nanobodies could be generated against SARS-CoV-2 viruses that have escaped H11-H4. Due to the limited resolution of the nanobody density in the cryo-EM map, the refined nanobody structure was replaced by the docked H11-H4–RBD crystal structure in the final model. 2a) and have shown some subtle differences in properties (Figs. The spike protein shows binding to CR3022 in the presence or absence of H11-H4. Data analysis was performed by generating a binding isotherm and best fit using the following parameters: n (number of sites), ΔH (calories per mole), ΔS (calories per mole per degree) and K (binding constant in mol−1). Extended Data Fig. (g–i) The three H11-D4 nanobodies with map contoured at 3.8 σ Chimera49. As a positive control, solutions with CR3022 333, 167, 84 and 42 nM were prepared. Comparison of the structures shows that the entire complex superimposes with a root-mean-square deviation (r.m.s.d.) 2a). These contacts have resulted in shifts of the RBD domains when compared to the non-complexed form13,26 (Extended Data Fig. 3a). Kim, A. S., Leaman, D. P. & Zwick, M. B. is funded by the EPA Cephalosporin Fund and The Townsend–Jeantet Charitable Trust (charity no. Adams, M. L., Katz, D. L. & Grandpre, J. Population-based estimates of chronic conditions affecting risk for complications from Coronavirus Disease, United States. e, H11-H4-Fc shows similar neutralization (ND50 = 4 nM) of live wild-type virus in a Vero cell-based assay in Oxford. Binding % = (X − min)/(max − min) × 100 where X = measurement of the competing component, min = buffer without binder biotinylated ACE2-Fc, max = biotinylated ACE2-Fc alone. Jakobi, A. J., Wilmanns, M. & Sachse, C. Model-based local density sharpening of cryo-EM maps. The surface on RBD that contacts H11-H4 is formed by Lys444 to Phe456 and Gly482 to Ser494 (Fig. The spike protein can only bind to ACE2 with the RBD in the ‘up’ state11 and this results in dissociation of the trimer. d, Loops CDR1, CDR2 and CDR3 of H11-H4 control recognition and are highlighted in magenta. 2b and Extended Data Fig. J. Mol. A serial half-log dilution (ranging from 1 μM to 0.1 nM) of analytes and controls was performed in a U-bottomed 96-well plate in a volume of 30 μl. This suggested the nanobody epitope overlaps with the ACE2 binding site on the RBD of spike. The following samples were injected: (1) a mixture of 1 µM nanobody H11-H4/H11-D4 and 0.1 µM RBD; (2) a mixture of 1 µM E08R (anti-Caspr2 Fab) Fab and 0.1 µM RBD; (3) 0.1 µM RBD; (4) a mixture of 1 µM nanobody H11-H4/H11-D4 and 0.1 µM spike; (5) a mixture of 1 µM E08R Fab and 0.1 µM spike; (6) 0.1 µM spike; (7) 1 µM nanobody H11-H4/H11-D4; (8) 1 µM E08R Fab. J.A.T., K.R.B., N.C., M.J.E., M.W.C., J.G.-J., M.L.K. d, RBD was bound by CR3022 (immobilized as CR3022-Fc on the chip). https://doi.org/10.1038/s41594-021-00566-w, https://doi.org/10.1016/j.chom.2020.06.010, https://doi.org/10.1101/2020.04.14.042010, https://doi.org/10.1101/2020.06.02.130161. Li, F., Li, W., Farzan, M. & Harrison, S. C. Structure of SARS coronavirus spike receptor-binding domain complexed with receptor. 6 Cryo-EM of the H11-D4–Spike complex. The response for the RBD H11-H4 mixture was larger, consistent with an H11-H4–RBD complex binding to CR3022. A pneumonia outbreak associated with a new coronavirus of probable bat origin. (b) As above, but with the Spike protein. ITC measurements were carried out using an iTC200 MicroCalorimeter (GE Healthcare) at 25 °C. }s�o;�7�zp�|vs�- 2b,e,f), blocks ACE2 binding (Figs. 1c,d). Acta Crystallogr. Li, F. Structure, function and evolution of coronavirus spike proteins. are funded by the Chinese Academy of Medical Sciences (CAMS) Innovation Fund for Medical Science (CIFMS), China (grant no. Cells (3 × 104 per well) were seeded the day before the assay on a flat-bottomed 96-well plate. The wells of microtiter plates (Greiner high and medium binding) were coated with 5 µg ml−1 neutravidin in PBS pH 7.4 overnight at 4 °C. Google Scholar. CAS  Superposition of the two complexes has confirmed that both nanobodies recognize the same epitope (Fig. Unfortunately, most of these antibodies do not cross-react with the SARS-CoV-2 RBD13. shown. CR3022 is shown as a positive control for this assay system and is similar to a previous report18. (f) Ribbon diagram of the complex and map (gray) contoured at 4 σ Chimera49. 9c,d). Ronco, C., Reis, T. & Husain-Syed, F. Management of acute kidney injury in patients with COVID-19. When RBD was pre-mixed with H11-H4, binding occurred with similar on and off rates, indicating that H11-H4 and CR3022 recognize different epitopes on RBD. Cell 181, 281–292 (2020). CompLife 3695, 163–174 (2005). Zhou, P. et al. In Figure 3d,e 5d % infectivity (% infectivity = 100 - % plaque reduction) is plotted against decreasing (left to right) concetration of the agent. Experiments were performed in duplicate with the mean ± s.d. designed the RBD construct for cell surface expression and carried out the cell-based ACE2 competition assays. J. Biol. J.H.N. World Health Organ. 2e,f), the discussion focuses on this variant, but, unless explicitly stated, is equally valid for H11-D4. Another antibody, which, like CR3022, does not block ACE2 binding but neutralizes the virus, has also been published34, but there are no further structural details. 4c). Detailed data plots are provided in Extended Data Fig. Data processing and refinement statistics are shown in Table 1. PubMed Central  Each experiment consisted of an initial injection of 0.4 μl followed by 16 injections of 2.4 μl of nanobody solution into the cell containing either spike or RBD, while stirring at 750 r.p.m. Structure 21, 1214–1224 (2013). Replicates and data for H11-D4 are provided in Extended Data Fig. a, Schematic of H11-H4-Fc, the IgG1 Fc (blue) fusion used in in vitro assays. Chem. Nanobody-Fc fusions showed neutralizing activity against SARS-CoV-2 (4–6 nM for H11-H4, 18 nM for H11-D4) and additive neutralization with the SARS-CoV-1/2 antibody CR3022. A single crystal of H11-H4–RBD–CR3022 was collected. Extended Data Fig. 5d). (a) Raw sensorgrams for the H11 parent nanobody. Model 51, 2778–2786 (2011). After the second round of panning, 93 individual clones were picked to inoculate 2xTYA and were grown overnight at 37 °C, while shaking at 250 r.p.m. Isolation and rapid sharing of the 2019 novel coronavirus (SARS-CoV-2) from the first patient diagnosed with COVID-19 in Australia. 4a and Extended Data Fig. The next day, the overnight culture was used to inoculate 2xTYA and infected with M13 helper phage to obtain clonal VHH-presenting phages. The mixture was then concentrated to 1 ml with a 5-kDa molecular weight cutoff (MWCO) concentrator and injected for gel filtration using a Superdex 200 10/300 system (GE) in 50 mM Tris pH 7, 150 mM NaCl. The spike protein is composed of S1 and S2 subunits. 3a,b and an experimental plate in Extended Data Fig. Preprint at bioRxiv https://doi.org/10.1101/2020.04.14.042010 (2020). The higher r.m.s.d. After washing, 100 μl of TMB substrate (SeraCare) was added and absorbance at 405 nm was measured with a microplate absorbance reader. 4h). The collected phages were amplified in exponentially growing TG1 Escherichia coli cells and plated on 2xTY agar plates supplemented with 100 μg ml−1 ampicillin. The direct injection of a nanobody has also shown promise in a mouse model of cobra venom intoxication39. Acta Crystallogr. Another study on CR3022 has reported highly effective SARS-CoV-2 neutralizing activity that appears to arise from destabilization of the spike trimer, a novel mechanism for neutralizing SARS-CoV-218. 1f. Data for H11-D4 are provided in Extended Data Fig. Proc. 3D classification was performed using emd_21374 low-pass-filtered to 60 Å. A SARS-like cluster of circulating bat coronaviruses shows potential for human emergence. The stoichiometry and thermodynamics of binding were characterized by isothermal titration calorimetry (ITC). 0 This drug was developed by Ablynx NV. Thank you for visiting nature.com. D Struct. Replicates and data for H11-D4 are provided in Extended Data Fig. Lancet Infect. Data acquisition and analysis were performed using the Origin scientific graphing and analysis software package (OriginLab). 211, 80–90 (2015). The antibody E08R (anti-Caspr2 Fab) was used as a negative control. 4e). Extended Data Fig. This assay yielded an IC50 of 34 nM for H11-H4-Fc, 28 nM H11-D4-Fc and 33 nM for VHH72-Fc25. General strategy to humanize a camelid single-domain antibody and identification of a universal humanized nanobody scaffold. 2c), plate assays (Fig. The epitope bound by VHH72 partly overlaps with the epitope bound by CR302218 (Extended Data Fig. b, Crystal structures of the H11-H4–RBD and H11-D4–RBD complexes were superimposed via the RBD (red), showing that both nanobodies recognize the same RBD epitope. The nanobodies were each incubated at room temperature with a purified prefusion-stabilized ectodomain of the SARS-CoV-2 spike protein13 (spike(trimer):nanobody = 1:4) and then vitrified on cryo-EM grids. Richard, G. et al. J. Infect. PLoS ONE 8, e54092 (2013). We have shown that H11-H4 binds with high affinity to RBD (Fig. Potent cross-reactive neutralization of SARS coronavirus isolates by human monoclonal antibodies. (c) The experimental plate with the codes noted below. Extended Data Fig. Data for H11-D4 are provided in Extended Data Fig. Biol. and W.J. Davis, I. W. et al. A highly conserved cryptic epitope in the receptor-binding domains of SARS-CoV-2 and SARS-CoV. Antibody-dependent enhancement of influenza disease promoted by increase in hemagglutinin stem flexibility and virus fusion kinetics. 7c). Contrast transfer function (CTF) estimation of full-frame non-weighted micrographs was performed using GCTF (v1.06), and non-template-driven particle picking was then performed within cryoSPARC (v2.14.1-live)47 followed by multiple rounds of two-dimensional (2D) classification. Extended Data Fig. Camelid VHH domains are highly conserved with their human counterparts, and their immunogenicity has been proposed to be low40, although humanization strategies are well developed41. All primers used in this work are listed in Supplementary Table 1. 1b) and this has been proposed to underlie the higher transmissibility of SARS-CoV-214. c, The superimposed nanobodies in b show a 7° pivot between H11-H4 and H11-D4. Plotkin’s Vaccines 8, 84–95 (2018). The grids were glow-discharged on low for 90 s (plasma cleaner PDC-002-CE, Harrick Plasma) to activate the nanowires. J.H., R.J.O. (c) FSC resolution criteria. Cells were washed with PBS and 50 μl of each mixture of ACE2 and an analyte was transferred to the cells and incubated for 1 h at room temperature. 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. Wrapp, D. et al. SARS-CoV-2 spike binds to ACE2 with a 10- to 20-fold higher affinity (KD of ~15 nM) than SARS-CoV-1 spike, a fact that has been proposed to drive its higher transmissibility13,31. S1 contains the RBD (highlighted in red). The reference flow cell was left blank. (d) 2Fo-Fc electron density map contoured at 2 σ for residues at the H11-D4–RBD interface. Additional sequence and structural changes between SARS-CoV-1 and SARS-CoV-2 (Tyr442→Leu455, Trp476→Phe490, Asn479→Gln493) combine to present a very different epitope and would seem to preclude cross-reactivity of H11-H4. Passive immunization. The plate was incubated at room temperature for 30 min and 0.5 ml of a single cell suspension of Vero E6 cells in D1 at 5 × 105 ml−1 was added. Frozen grids were first screened on a Glacios microscope operating at 200 kV (Thermo Fisher Scientific) before imaging on a Titan Krios G2 (Thermo Fisher Scientific) at 300 kV. The higher affinity results from sequence changes in RBD (Fig. volume 27, pages 846–854 (2020)Cite this article, An Author Correction to this article was published on 03 February 2021, An Author Correction to this article was published on 15 October 2020. However, we noted that in the nanobody–spike complex, the ‘up’ RBD (subunit A) makes contacts with the nanobody that is bound to ‘down’ RBD (subunit C) (Extended Data Fig. Superposition of the RBD–ACE2 complex29,30 on the H11-H4–RBD complex reveals that H11-H4 would, consistent with biophysics (Fig. 2e). Such additive combinations are a well-known strategy to reduce the propensity of the virus to escape by mutating. Liebschner, D. et al. When H11-H4 is bound to RBD, it would prevent ACE2 binding due to steric clashes. A topology diagram shows that the nanobody is composed of two β-sheets. The ability of these constructs to block ACE2 binding to RBD was tested in two assays. Antibody dilutions were run in duplicate and an internal positive control for the PRNT assay was also run in duplicate using a sample of heat-inactivated (56 °C for 30 min) human MERS convalescent serum known to neutralize SARS-CoV-2 (National Institute for Biological Standards and Control, UK). PubMed Google Scholar. To determine the binding affinity of nanobody H11 for the SARS-CoV-2 RBD, RBD-Fc was immobilized onto the sample flow cell of the sensor chip. 3c. Electron densities for both complexes are shown in Extended Data Fig. Emerg. Methods 14, 290–296 (2017). Motion correction and alignment of 2× binned super-resolution movies was performed using Relion (v3.1)46 with a 5 × 5 patch-based alignment. Proc. Peyvandi, F. et al. Approximately 6 nl of the complex were applied to the grids using a Chameleon EP system (SPT Labtech) at 81% relative humidity and ambient temperature. Data are presented as mean and s.d. The structure of the loop centered at Val483 of the RBD has changed upon binding of H11-H4 (Extended Data Fig. Cells (3 × 104 per well) were seeded the day before the assay on a flat-bottomed 96-well plate. Caly, L. et al. (1) ---- … Diffraction data were also collected and processed at beamline I03 at Diamond Light Source. The plaques caused by the virus are visible. 303 0 obj <>stream Huo, J., Le Bas, A., Ruza, R.R. J. Comput. J.H. the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in All assays were performed using a Sensor Chip Protein A (GE Healthcare), with a running buffer of PBS pH 7.4 supplemented with 0.005% vol/vol surfactant P20 (GE Healthcare) at 25 °C. PubMed  Acta Crystallogr. For our in vitro binding assays (Fig. 9 H11-H4 and VHH72 recognize different epitopes. The coordinates from the spike–H11-D4 structure were rigid-body-docked into the spike–H11-H4 cryo-EM density in Chimera49 and then refined with multiple rounds of jelly body refinement using RefMac5 via CCP-EM GUI53,54, and manual intervention with Coot resulted in a final correlation coefficient of 0.78. Science 368, 630–633 (2020). Med. In the first assay, MDCK-SIAT1 cells stably expressing human ACE2 (MDCK-ACE2) were seeded on plates and the ability of various analytes (H11-H4-Fc, H11-D4-Fc, ACE2-Fc, CR302218 and VHH72-Fc) to block binding of RBD was measured (Fig. 2d and Extended Data Fig. We suggest the additional interactions are responsible for this observation and for the higher enthalpy and greater entropic penalty observed for nanobody binding to spike when compared to RBD (Fig. Mammalian, including human, antibodies generally have two chains (heavy and light), but camelids, in addition to two-chain antibodies, also possess a single-heavy-chain antibody variant21. 2e,f). 130, 2757–2765 (2020). The model was constructed by superimposing the H11-H4–RBD complex onto the EM structure of the closed form of Spike (PDB 6vxx13). Vincke, C. et al. Other sequence differences are in black text, with conservative substitutions indicated by colons (:) underneath. Afterwards, the virus-antibody mixture was transferred into the wells of a twice Dulbecco’s PBS-washed 24-well plate containing confluent monolayers of Vero E6 cells (ECACC 85020206, PHE) that had been cultured in MEM containing 10% (vol/vol) FBS. 35, W375–W383 (2007). (c) H11-H4-Fc. 2d and Extended Data Fig. Crystallogr. Biol. All authors analyzed the data. The chimeric fusions were tested in a plaque reduction neutralization test at the Public Health England Laboratory for SARS-CoV-2 virus, and showed an ND50 of 6 nM for H11-H4-Fc (95% CI 3–9 nM) and ND50 of 18 nM for H11-D4-Fc (95% CI 9–68 nM) (Fig. The H11-H4 and H11-D4 nanobodies may find application in a cocktail of laboratory-synthesized neutralizing antibodies given for passive immunization of severely ill COVID-19 patients. We thank colleagues at the SGC (Oxford) mass spectrometry service for their assistance. Cell-based competition assays used MDCK-SIAT1 cells derived from a commercial source (Sigma-Aldrich). 3). Phaser crystallographic software. 8a). The effectiveness of convalescent plasma and hyperimmune immunoglobulin for the treatment of severe acute respiratory infections of viral etiology: a systematic review and exploratory meta-analysis. Some, but not all, of these nanobodies blocked ACE2 binding and no molecular insights into their mode of action were reported35. https://doi.org/10.1038/s41594-020-0469-6, Comparative Analysis of Antigen-Specific Anti–SARS-CoV-2 Antibody Isotypes in COVID-19 Patients, Recent biotechnological advances as potential intervention strategies against COVID-19, Bispecific VH/Fab antibodies targeting neutralizing and non-neutralizing Spike epitopes demonstrate enhanced potency against SARS-CoV-2, Biosensing Detection of the SARS-CoV-2 D614G Mutation, Identification of nanobodies against hepatocellular carcinoma marker glypican-3. Huo, J. et al. Wrapp, D. et al. Excess liquid was removed by blotting for 6 s with a blotting force of −1 using Vitrobot filter paper (grade 595, Ted Pella) at 4.5 °C and 100% relative humidity. SPHIRE-crYOLO is a fast and accurate fully automated particle picker for cryo-EM. CTF estimation of full-frame non-weighted micrographs was performed using GCTF (v1.06) and non-template-driven particle picking was then performed within crYOLO52 followed by 2D classification. Using the RBD, the trimeric spike molecule binds to ACE2 on human cells (a single ACE2 is shown in blue). Similar results were observed using spike protein instead of RBD. Graphs were plotted as percent binding of biotinylated RBD to ACE2. wrote the manuscript with input from all authors. Enrichment after each round of panning was determined by plating the cell culture with 10-fold serial dilutions. Three loops—complementarity-determining region 1 (CDR1), CDR2 and CDR3—control antigen binding and are highlighted in purple. Preprint at bioRxiv https://doi.org/10.1101/2020.06.02.130161 (2020). In vivo neutralization of α-cobratoxin with high-affinity llama single-domain antibodies (VHHs) and a VHH-Fc antibody. Experiments were performed in duplicates with mean ± s.d. The best 2D classes clearly showing details consistent with the spike complex were selected for further processing. 16). ������8��k0@�EG3P� $m$8����� R�O i � ���,���3�fae�c>���|�� ��"�vK�~.=6=E�;����N�i@��� w��: v&�{ (a) Unbiased 2D class averages of the complex. Around 33% of those admitted to UK hospitals with COVID-19 have died6. Walls, A. C. et al. J. Virol. Using a random mutagenesis approach, we identified two affinity matured mutants, H11-D4 and H11-H4, which differ from H11 and each other at five residues within CDR3 (Figs. This region of SARS-CoV-2 RBD has several sequence changes when compared to SARS-CoV-1 RBD (Fig. USA 96, 9459–9464 (1999). The resulting mixtures were analyzed as described above in triplicate experiments and the wells are shown in Extended Data Fig. Crystallogr. (d) H11-H4-Fc varied and CR3022 held constant at 84 nM. D Biol. Biol. 4a. PubMed  Initially the data were processed as C3 but relaxed to C1 as the RBD and nanobody densities were poor. 1 Biophysics of the nanobody binding to RBD. The two helices and turn of ACE2 that contact RBD are shown in cartoon representation and are colored in light blue. The IC50 values of the nanobodies against ACE2 were determined using nonlinear regression [inhibitor] versus normalized response curve fit using GraphPad Prism 8. In the second competition assay, analytes (H11-H4-Fc, H11-D4-Fc, ACE2-Fc, CR302218, VHH72-Fc25) were assessed for their ability to block ACE2 binding to MDCK cells that expressed RBD on their surface (Fig. b, Biotinylated RBD was mixed with analytes at various ratios and then added to MDCK-SIAT1 cells stably expressing human ACE2. Murshudov, G. N. et al. 7a) are indistinguishable given their resolution (Table 1). 4d). 4a–c. 2, 218 (2019). Biol. 14, e1007236 (2018). Dashed lines are 50% and 10% of the control, respectively. Given the very high degree of similarity between the complexes, we again focused the description on the H11-H4–RBD complex (Fig. 9a). Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Get the most important science stories of the day, free in your inbox. 282 0 obj <>/Filter/FlateDecode/ID[<17EBF81033552C45AF708308C9205D44>]/Index[264 40]/Info 263 0 R/Length 96/Prev 1138682/Root 265 0 R/Size 304/Type/XRef/W[1 3 1]>>stream EM results were obtained at the cryo-EM facility (OPIC) in the Division of Structural Biology, University of Oxford, part of the UK Centre of Instruct-ERIC, and the national EM facility at Diamond, eBIC, through rapid access proposal BI27051. The ligated vector was transformed into TG1 cells by electroporation to give a phage library consisting of ~2 × 109 independent clones. CR3022 was used as a positive control, and under these conditions an ND50 of 93 nM was observed, similar to a previous report18 (Fig. Interestingly the contact surface of H11-H4 on RBD shows only a small overlap with the ACE2 contact surface (Figs. Crystals were soaked in cryoprotectant containing 70–75% reservoir solution and 20–25% glycerol for a few seconds, then mounted in loops and frozen in liquid nitrogen before data collection at beamline I03 of Diamond Light Source, UK. Salje, H. et al. Statistics for X-ray data collection and structure refinement are provided in Table 2. The use of convalescent serum has shown clinical promise in patients severely ill with SARS-CoV37 and most recently SARS-CoV-29; such passive immune therapy has a long history in medicine38. Chem. 2d and 5c), we investigated a combination of H11-H4 and CR3022 (CR3022 concentration fixed at 84 nM). The spike protein comprises an N-terminal (S1) subunit, which contains the roughly 200-residue receptor binding domain (RBD)10,11, and a C-terminal subunit (S2), which contains the fusion protein12 (Fig. This is due to van der Waals clashes, principally between regions of H11-H4 that are not in contact with the RBD and regions of ACE2 (also not in contact with RBD; Fig. Structural Biology, The Rosalind Franklin Institute, Harwell Science & Innovation Campus, Didcot, UK, Jiandong Huo, Maud Dumoux, Miriam Weckener, Lucile Moynié, Raymond J. Owens & James H. Naismith, Division of Structural Biology, University of Oxford, The Wellcome Centre for Human Genetics, Headington, Oxford, UK, Jiandong Huo, Audrey Le Bas, Reinis R. Ruza, Helen M. E. Duyvesteyn, Tomas Malinauskas, Philip N. Ward, Jingshan Ren, Daming Zhou, Peter J. Harrison, Yuguang Zhao, Loic Carrique, Pranav N. M. Shah, David I. Stuart, Raymond J. Owens & James H. Naismith, Protein Production UK, The Rosalind Franklin Institute – Diamond Light Source, The Research Complex at Harwell, Harwell Science & Innovation Campus, Didcot, UK, Jiandong Huo, Audrey Le Bas, Philip N. Ward, Peter J. Harrison, Raymond J. Owens & James H. Naismith, Diamond Light Source Ltd, Harwell Science & Innovation Campus, Didcot, UK, Halina Mikolajek, Daniel K. Clare, Vinod K. Vogirala, Julika Radecke & David I. Stuart, MRC Human Immunology Unit, Weatherall Institute of Molecular Medicine, University of Oxford, John Radcliffe Hospital, Oxford, UK, Tiong Kit Tan, Pramila Rijal & Alain R. Townsend, Centre for Translational Immunology, Chinse Academy of Medical Sciences Oxford Institute, University of Oxford, Oxford, UK, Sir William Dunn School of Pathology, University of Oxford, Oxford, UK, Javier Gilbert-Jaramillo, Michael L. Knight & William James, National Infection Service, Public Health England, Porton Down, Salisbury, UK, Julia A. In addition, Arg52 makes hydrogen bonds with the main-chain Ser103 (side chain omitted) and Tyr109. The experimental plates are shown in Extended Data Fig. f, ITC measurements show a KD of 44 ± 3 nM and a 1:1 ratio for association between spike protein and H11-H4. 2c). and P.J.H. 1e). 1b and 5b). Cell 181, 271–280 (2020). H11-H4 residues are colored as in e. RBD residues are colored as in f. The region of the RBD in contact with the nanobody is ordered in the nanobody complex but is disordered in the EM prefusion stabilized holo spike structures (PDB 6VSB, 6VYB and 6VXX)13,26, precluding detailed analysis. Nat. and A.R.T. (d) Particle orientation distribution for the final map showed no preferred orientation. (e) Final map colored according to local resolution. 4d,e). 5a). Bull. Either the nanobodies have bound equally well to all conformational states present or the equilibration between these states was faster than the binding event. 5a). The script used in R was based on a previously reported source script44. PDB 6ZH9 (H11-H4–RBD–CR3022), 6ZBP (H11-H4–RBD), 6YZ7 and 6Z2M (H11-D4–RBD–CR3022) and 6YZ5 (H11-D4–RBD). 5, 6 and 7a). The lack of conservation of the H11-H4 epitope between SARS-CoV-1 and SARS-CoV-2 raises the possibility that SARS-CoV-2 variants may emerge that retain ACE2 receptor binding but are no longer recognized by H11-H4 or its relatives. and D.I.S. ACE2-Fc was biotinylated as above. 9, 382–385 (2020). h�b```e``re`a``6bb@ !�+sl r�C\"_4Lz��=��ü j\VL��V�,`���s���g/_��*�(��Y�� �V^��ޱ$,--Md$�k�v�Kݸ��xʵc��2�n0�S���}��2J�L�! Two studies have reported crystal structures of CR3022 bound to SARS-CoV-2 RBD and show that the target epitope is distant from the ACE2 binding region17,18, which is consistent with the observation that CR3022 does not block RBD binding to ACE2. Maps in f-i used amplitudes scaled based on the refined coordinates using LocScale63. EP/S025243/1. Structural basis for the recognition of SARS-CoV-2 by full-length human ACE2. Correspondence to A second form appeared later and yielded 2.7 Å from a single crystal, although the data were anisotropic. Nanobody sequences are provided in Supplementary Note 2. Purified spike protein in 10 mM HEPES, pH 8, 150 mM NaCl was incubated with H11-H4 purified in 50 mM Tris, pH 7, 150 mM NaCl, at a molar ratio of 1:3.6 (spike trimer:nanobody) at 16 °C overnight. Cells were then washed with PBS and incubated for 1 h with the second-layer streptavidin-HRP (S911, Life Technologies), diluted to 1:1,600, and developed as above. 7b)—contacts that are absent in the holo spike. 1 and 2, Supplementary Notes 1 and 2 and Supplementary Table 1. PubMed  This would suggest that neutralization of the virus, even at a relatively late stage in the disease, may be a useful COVID-19 therapy. 8c,d. D Struct. Elife 6, e27131 (2017). We performed an SPR-based competition assay in which ACE2-Fc was immobilized and then binding of RBD was monitored in the presence or absence of H11-H4 or H11-D4. The Pro469–Pro470 turn in the SARS-CoV-1 RBD structure33 is very different to the structure at Val483–Glu484 in SARS-CoV-2. CAS  Structure, function and antigenicity of the SARS-CoV-2 spike glycoprotein. T.K.T. Crystallogr. The amount of biotinylated RBD bound was measured. Each experiment was performed in triplicate in a 24-well tissue culture plate. Cell 181, 1436–1441 (2020). 3b,c) and neutralization experiments (Fig. Commun. by the UK Medical Research Council (MR/N00065X/1). Tree, Karen R. Buttigieg, Naomi Coombes, Michael J. Elmore & Miles W. Carroll, You can also search for this author in a, Maturation by mutagenesis of CDR3 region of H11 resulted in H11-D4 and H11-H4.
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