Unexpected Receptor Functional Mimicry Elucidates Activation of Coronavirus Fusion

By A. Walls, X. Xiong, Young-Jun Park, M. A. Tortorici, Joost Snijder, J. Quispe, E. Cameroni, R. Gopal, M. Dai, A. Lanzavecchia and 4 more. 2020

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a structural framework for understanding coronavirus neutralization by human antibodies and shed light on activation of coronavirus membrane fusion, which takes place through a receptor-driven ratcheting mechanism. glycine immediately neutralized using transfection puriﬁed afﬁnity CH1-XL HiTrap Relion3.0 (Kimanius et al., 2016) with a box size of 768 pixels 2 binned to 384 pixels 2 yielding a pixel size of 1.37A˚. Reference free 2D classiﬁcation was used to parse 240,000 particles from the original 500,000 particle images using Relion3.0. An initial model was generated from PDBID 5W9J with all MERS-CoV S domain B closed and removing the G4 Fab (Pallesen et al., 2017). Relion3.0 3D classiﬁcation with C1 symmetry was utilized to select the (cid:1) 126,000 best particles for state 1 and the (cid:1) 114,000 best particles for state 2. CTF reﬁnement in Relion3.0 was used to reﬁne per-particle defocus values. Particles images from each state were subjected to the Bayesian polishing procedure implemented in Relion3.0 (Zivanov et al., 2018, 2019) and 3D reﬁnement before performing 3D classiﬁcation, without reﬁning angle and shifts, using soft masks focusing on the B domains and Fabs with a tau factor of 50. 82,000 and 78,000 particles were selected for states 1 and 2, respectively. Particle images from state 1 underwent another round of domain B/LCA60 focused classiﬁcation to select 54,000 particles used for the ﬁnal reconstruction. Finally, CryoSPARC non-uniform reﬁnement (Punjani et al., 2017) was used to obtain the ﬁnal reconstructions at 3.5 and 3.6 A˚ resolution, respectively, using soft masks excluding the open B domains and bound Fabs. Reported resolutions are based on the gold-standard FSC = 0.143 criterion (Rosenthal and Henderson, 2003; Scheres and Chen, 2012) and Fourier shell correlation curves were corrected for the effects of soft masking by high-resolution noise substitution(Chen et al., 2013). Local resolution estimation, ﬁltering and sharpening was carried out using CryoSPARC. Analysis was carried out using the state 1 structure since the reconstruction is best resolved. 0.1 M HEPES pH 7.0. LCA60 dataset was ALS beamline 5.0.1 and processed to 3.0 A˚ resolution space group P2 dimensions 70.72 A˚ b 69.57 A˚ and c = 93.84 A˚, XDS 2010) and Aimless Fab S230 crystals were in hanging drop set up with a mosquito at 20 (cid:3) C 150 nL protein and 150 nL mother liquor solution containing 3.5 M sodium formate pH 7.0. Crystals were cryo-protected using the mother liquor solution supplemented with 30% glycerol. The S230 dataset was collected at the ALS beamline 5.0.1 and processed to 1.5 A˚ resolution in space group P2 1 2 1 2 1 using XDS and Aimless. The structures of Fab LCA60 and Fab S230 were solved by molecular replacement using Phaser (McCoy et 2018) and homology models as search models. The coordinates were subsequently improved and completed using Buccaneer (Cowtan, 2006) and COOT (Brown et Emsley et and reﬁned with BUSTER-TNT (Blanc 2004) and REFMAC5 (Vagin 2004). Simu-lated annealing and omit map calculations were performed Phenix (Adams et al., 2010). The quality of the crystal structures were analyzed using MolProbity al., 2010). data m g of S for 4-14 hr in 50mM sodium acetate pH 4.4 at 37 (cid:3) C in a 20 m L reaction. 6 m g of S were then incubated in 100mM Tris pH 8.5, 2% sodium deoxycholate, 10mM tris(2-carboxyethyl)phosphine, and 40mM iodoacetamide at 95 (cid:3) C for ﬁve minutes and 25 (cid:3) C for thirty minutes in the dark. Denaturated, reduced, and alkylated S (1.6 m g) was then diluted into fresh 50mM Ammonium bicarbonate and incubated for 14 hr at 37 (cid:3) C either with 0.032 m g of trypsin (Sigma Aldrich), chymotrypsin (Sigma Aldrich), trypsin then sequentially chymotrypsin, alpha lytic protease (Sigma), or AspN protease (Pierce). Formic acid was then added to a ﬁnal concentration of 2% and the samples spun at 14,000 rpm for 20 minand then again for 5 min to precipitate the sodium deoxycholate and collect the peptides from the supernatants. The SARS S digests with trypsin, chymotrypsin and alpha lytic protease were processed in parallel after cotton-enrichment of glycopeptides. Approximately 0.5 mg of cotton wool was packed into 100 m L pipette tips. Glycopeptides were diluted in 80% acetonitrile with 0.1% formic acid and bound to the cotton plug, followed by washing of the cotton plug with 80% acetonitrile with 0.1% formic acid. Glycopeptides were eluted in 0.1% formic acid in water. resolution setting of 30,000 with an AGC target of 1*10 5 was used for MS2. The data was then analyzed using Byonic (Bern et al., 2012) against a custom database of recombinant coronavirus spike proteins, searching for glycan modiﬁcations with 12/24 ppm search windows for MS1/MS2, respectively. The in-solution digestions of the recombinant ectodomain were searched against a custom database of recombinant coronavirus spike proteins, proteases, and common contaminants. The in-gel digestion experiments were ﬁrst searched against a database comprised of the full MERS-CoV proteome and the full Chlorocebus sabaeus (virus produced in VeroE6 cells) or human proteome (ectodomain produced in HEK293F cells) retrieved from UniProt, with variable N-linked glycosylations omitted from the search to identify background contaminants. The top 16 contaminants were then assembled together with the S sequence into a focused database for the glycopeptide identiﬁcations. In all experiments, up to three missed cleavages were permitted using C-terminal cleavage at R/K for trypsin, F/Y/W/M/L for chymotrypsin, R/K/F/Y/W/M/L for trypsin-chymotrypsin, T/A/S/V for alpha lytic protease, and N-ter-minal cleavage at D for AspN. Carbamidomethylation of cysteine was set as ﬁxed modiﬁcation, methionine oxidation and carbamidomethylation of lysine as variable common 1, glycan modiﬁcations as variable common 2, with a variable common max. parameter of 3. The N-linked glycan databases of Byonic were merged into a single non-redundant set of glycan compositions for the searches. All reported glycopeptides in the results ﬁles were manually inspected quality of fragment assignments. All glycopeptide identiﬁcations sequon. Glycans search ﬁle search pooled on the number of HexNAc, Fuc or NeuAc residues distinguish high-mannose/hybrid/complex glycosylation, fucosylation and sialylation, respectively. We recently discovered that the puriﬁed ACE2 ectodomain used in the manuscript encompasses residues 19–741 of macaque ACE2 instead of residues 1–615 of human ACE2. Although the binding interface is conserved, the presence of the endogenous ACE2 dimer-ization domain affects the afﬁnities reported in Figure S7 due to avidity. New measurements were carried out with the human ACE2 ectodomain (residues 19–615), and the corresponding binding afﬁnities for the RBD were corrected (Starr, T.N., et al. (2020). Deep Mutational Scanning of SARS-CoV-2 Receptor Binding Domain Reveals Constraints on Folding and ACE2 Binding. Cell 182, 1295– 1310. 10.1016/j.cell.2020.08.012). Our conclusions remain unchanged. We apologize for any confusion that this error may have caused.

Subfields: Immunology, Infectious Diseases, Radiology, Nuclear Medicine and Imaging

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Citations in the corpus

Molecular Architecture of the SARS-CoV-2 Virus (2020, neutral): “, 2020a), a feature shared with Middle East Respiratory Syndrome (MERS) and SARS-CoV (Walls et al., 2019).”
Nanobody cocktails potently neutralize SARS-CoV-2 D614G N501Y variant and protect mice (2021, neutral): “Antiviral therapies against human coronaviruses that block receptor engagement and the ability to undergo membrane fusion will effectively inhibit the process of viral entry and block infection (25).”
Deep mutational scanning of SARS-CoV-2 receptor binding domain reveals constraints on folding and ACE2 binding (2020, positive): “To better define the RBD’s evolutionary capacity for antibody escape, we examined mutational constraint in the epitopes of antibodies with available structures that bind the SARS-CoV-1 or SARS-CoV-2 RBD (Figures 7A, S7A,B) (Hwang et al., 2006; Pak et al., 2009; Pinto et al., 2020; Prabakaran et al.,”
Structural and Functional Analysis of the D614G SARS-CoV-2 Spike Protein Variant (2020, positive): “Models of coronavirus S-mediated membrane fusion describe ACE2 binding to all three RBD domains in the open conformation as destabilizing the pre-fusion S trimer, leading to dissociation of S1 from S2 and promoting transition to the post-fusion conformation (Pallesen et al., 2017; Walls et al., 2019”
Characterization of neutralizing antibodies from a SARS-CoV-2 infected individual (2020, background): “High resolution structures of the SARS-CoV-2 prefusion-stabilized spike (S) ectodomain revealed that it adopts multiple conformations with either one receptor binding domain (RBD) in the “up” or “open” conformation or all RBDs in the “down” or “closed” conformation, similar to previous reports on bo”
Structure, Function, and Antigenicity of the SARS-CoV-2 Spike Glycoprotein (2020, methodology): “, 2015) in complex with SARS-CoV S and MERS-CoV S to provide molecular-level information of the mechanism of competitive inhibition of S attachment to the host receptor (Walls et al., 2019).”
The deadly coronaviruses: The 2003 SARS pandemic and the 2020 novel coronavirus epidemic in China (2020, neutral): “During the binding of the subunit S1 to its cognate receptor, it is important to note that the S1 subunit exists in 2 different states, a "down" conformation and an "up" conformation state, which corresponds to a receptor-inaccessible state and an unstable receptor-accessible state, respectively [12”
Insights into SARS-CoV-2 genome, structure, evolution, pathogenesis and therapies: Structural genomics approach (2020, positive): “Because of the vital function of the S protein, it is considered as an attractive target for antibody-mediated vaccine and drug development [62,73].”
Coronavirus disease 2019 (COVID-19): current status and future perspectives (2020, background): “Binding of the S1 subunit to a host receptor can destabilise the prefusion trimer, leading to shedding of the S1 subunit and transition of the S2 subunit into a highly stable postfusion conformation [18] .”
Structural insights into coronavirus entry (2019, background): “…MERS-CoV S structures in complex with neutralizing antibodies isolated from survivors showed both antibodies com- petitively blocked receptor interaction, in agreement with previous surface plasmon resonance data (Corti et al., 2015; Rockx et al., 2008; Traggiai et al., 2004; Walls et al., 2019).”
SARS-CoV-2 Spike protein variant D614G increases infectivity and retains sensitivity to antibodies that target the receptor binding domain (2020, positive): “Previous structural studies of SARS-CoV and MERS-CoV S proteins identified similar conformations, with only MERS-CoV S protein exhibiting an all open state like that reported here (Gui et al., 2017; Pallesen et al., 2017; Walls et al., 2019; Yuan et al., 2017).”
A highly conserved cryptic epitope in the receptor-binding domains of SARS-CoV-2 and SARS-CoV (2020, background): “15 53 (sequences from PDB 6NB6 (38)), the heavy-chain and light-chain plasmids were 54”