key: cord-0948815-x2v6238q
authors: Zhu, Xing; Mannar, Dhiraj; Srivastava, Shanti S.; Berezuk, Alison M.; Demers, J. -P.; Saville, James W; Leopold, Karoline; Li, Wei; Dimitrov, Dimiter S.; Tuttle, Katharine S.; Zhou, Steven; Chittori, Sagar; Subramaniam, Sriram
title: Cryo-EM Structure of the N501Y SARS-CoV-2 Spike Protein in Complex with a Potent Neutralizing Antibody
date: 2021-01-12
journal: bioRxiv
DOI: 10.1101/2021.01.11.426269
sha: b8f99d60bb24db5a93c0e99df1ac20d1d7f526d6
doc_id: 948815
cord_uid: x2v6238q

The recently reported “UK variant” of SARS-CoV-2 is thought to be more infectious than previously circulating strains as a result of several changes, including the N501Y mutation. Here, we report cryo-EM structures of SARS-CoV-2 spike protein ectodomains with and without the N501Y mutation, in complex with the VH fragment of the potent neutralizing antibody, VH -Fc ab8. The mutation results in localized structural perturbations near Y501, but VH -Fc ab8 retains the ability to bind and neutralize pseudotyped viruses expressing the N501Y mutant with efficiencies comparable to that of unmutated viruses. Our results show that despite the higher affinity of ACE2 for the N501Y mutant, it can still be neutralized efficiently by an antibody that binds epitopes in the receptor binding domain of the SARS-CoV-2 spike protein.

The rapid international spread of SARS-CoV-2 is associated with numerous mutations that alter viral fitness. Mutations In December 2020, new variants of SARS-CoV-2 carrying several mutations in the spike protein were documented in the UK (SARS-CoV-2 VOC202012/01) and South Africa (501Y.V2) (14, 15) . Early epidemiological and clinical findings have indicated that these variants show increased transmissibility in the population (16) . Despite being phylogenetically distinct, a common feature of both UK and South African variants is the mutation of residue 501 in the RBD from Asn to Tyr (N501Y). X-ray crystallography and cryo-EM structural studies have identified N501 as a key residue in the interaction interface between RBD and ACE2. N501 is involved in critical contacts with several ACE2 residues (5, 6, 10, 13) . Studies carried out in a mouse model before the identification of the new UK variant have suggested that mutations of residue 501 could be linked to increased receptor binding and infectivity (17, 18) . Understanding the impact of N501Y on antibody neutralization, ACE2 binding, and viral entry is therefore of fundamental interest in the efforts to prevent the spread of COVID-19.

We recently described the potent neutralization of SARS-CoV-2 with VH -Fc ab8, an antibody derived from a large human library of antibody sequences (19) . To understand the effects of ab8 binding on the N501Y mutant, we expressed and purified spike protein ectodomains with and without the N501Y mutation in Expi293 cells. A cryo-EM structure of the spike protein ectodomain with the N501Y mutation was obtained at an average resolution of ~ 2.8 Å ( Figure   S1 ). The structure shows no significant global changes in secondary or quaternary structure as a result of the mutation when compared to the previously published structure of the spike protein ectodomain with an Asn residue at position 501 (referred to as the "unmutated" form; Figure S2 ).

Cryo-EM structural analysis of the complex formed between VH ab8 and the unmutated spike protein ectodomain shows two distinct quaternary states, both at average resolutions of 2.4 Å (Figures 1, S3 ). One class ( Figure 1A ) has two RBDs in the "down" position both bound to VH ab8, with the third in a predominantly "up" position, but with weaker density for both the RBD and the bound VH ab8 fragment, suggesting that this domain is flexible relative to the rest of the ectodomain. In the second class ( Figure 1B) , one of the RBDs is in the down position, with two in the up position, each bound to VH ab8. Global refinement results in lower resolutions for the RBD regions relative to the rest of the spike, but local refinement of just the RBD-VH ab8 regions results in structures with local resolutions better than ~ 3 Å in each case ( Figure S3 ). The interface between the RBD and VH ab8 is well-defined with key interactions at the interface mediated by residues in the stretch between V483 and S494, along with a few other interactions contributed by non-contiguous RBD residues ( Figures 1C, 1D ). Residue 501 of the spike protein RBD is at the periphery of the footprint of ab8, with minimal, if any, interactions with the antibody.

The N501Y trimeric spike protein ectodomain shows a single dominant conformation ( Figure   2A ) with the same distribution as in the first class presented for the complex with unmutated spike ectodomain ( Figure 1A ). Two VH ab8 fragments are bound to RBDs in the down conformation, with weak density for the other RBD, which is flexible and primarily in the up position. The global average resolution of the map is ~ 2.8 Å, with lower local resolution in the RBD regions, but local refinement yields maps of the VH ab8-RBD interface at resolutions better than ~ 3 Å ( Figure S4 ). Cryo-EM density maps unambiguously show the location of residue 501 in both unmutated and N501Y mutant spike protein ectodomains ( Figures 2B, C) . The presence of the mutation does not significantly alter any of the interactions between the RBD and VH ab8 or the overall conformation of the loop ( Figure S5 ). However, replacement of the asparagine residue by the bulkier tyrosine side chain results in subtle local rearrangements, noticeably in the orientations of Y505 and Q498 ( Figures 2C, 2D) .

Comparison of the structures reported here with those reported for the ACE2-RBD complex from earlier X-ray crystallography and cryo-EM studies enable visualization of the similarities and differences in the modes of binding ( Figure 3 ). ACE2 binding has been observed only to RBDs in the up position, likely because of steric constraints in accommodating ACE2 in the down conformation. In contrast, the much smaller VH ab8 fragment binds the RBD both in up and down positions. Despite these differences, and the fact that ACE2 and VH ab8 each have distinctive directions of approach in their contact with the RBD, there is a good match in the RBD footprint between VH ab8 and ACE2, accounting for the potent neutralization by the VH -Fc antibody (19) . Residue 501 is located towards the outer edge of the contact zone between RBD in the ACE2 complex and just outside the zone of contact of VH ab8 with RBD, providing a structural rationale for the findings we describe here on the minimal effect of the N501Y mutation on interaction with the antibody.

We next carried out a series of experiments to test the effects of the N501Y mutation on ACE2 binding and the relative strengths of binding and neutralization potency of VH -Fc ab8 ( Figure 4 ). Competition experiments establish that ACE2 can displace VH-Fc ab8 bound to unmutated and N501Y mutant spike proteins with similar efficiency ( Figure 4B ). Consistent with these measurements, neutralization experiments carried out with VH-Fc ab8 shows that it is able to neutralize the N501Y mutant with a potency similar to that of the unmutated form ( Figure 4C ).

The neutralization studies were further confirmed by ELISA measurements of relative binding efficiency VH-Fc ab8. As shown in Figure 4D , the N501Y mutation has no significant effect on VH-Fc ab8 binding. Finally, negative stain studies of the complex formed between trimeric spike proteins and soluble ACE2 show that the N501Y mutant has a higher stoichiometry of ACE2 binding in the presence of similar concentrations of ACE2 ( Figures 4E, F) . Collectively, the binding, neutralization, and electron microscopic analyses show that the N501Y mutation results in increased efficiency of ACE2 binding, but has relatively minor effects on binding and potency of an antibody that targets neutralizing epitopes in the RBD.

Our studies with the N501Y mutant confirm the expectation that the rapid spread of the UK variant of SARS-CoV-2 is likely due to the viruses being more infectious. While there can be multiple origins for the increased infectivity, our biochemical and structural studies establish that the N501Y mutation results in increased ACE2 binding efficiency. The competition assays with a strongly neutralizing antibody show that it competes for binding with the spike trimer-ACE2 interaction in a concentration-dependent manner. Our results suggest that despite the higher infectivity of SARS-CoV-2 viruses carrying the N501Y mutation, the availability of the extended epitope surface on the RBD will likely enable effective neutralization by antibodies elicited by immunization with vaccines that are currently in production. With the continued spread of SARS-CoV-2, it appears likely that further mutations that enhance viral fitness will emerge. Cryo-EM methods to rapidly identify footprints of antibodies that are generated by current and future generations of vaccines could thus add a critical tool to the arsenal of efforts to prevent and treat COVID-19. 

The wild type SARS-CoV-2 S HexaPro expression plasmid was a gift from Jason McLellan (7) and obtained from Addgene (plasmid #154754; http://n2t.net/addgene:154754; RRID:Addgene_154754). The The protein was eluted with elution buffer (20 mM Tris pH 8.0, 500 mM NaCl, 500 mM imidazole). Elution fractions containing the protein were pooled and concentrated (Amicon Ultra 100 kDa cut off, Millipore Sigma) for gel filtration. Gel filtration was conducted using a Superose 6 10/300 GL column (Cytiva) pre-equilibrated with GF buffer (20 mM Tris pH 8.0, 150 mM NaCl). Peak fractions corresponding to soluble protein were pooled and concentrated to 4.5 -5.5 mg/mL (Amicon Ultra 100 kDa cut off, Millipore Sigma). Protein purity was estimated as > 95% by SDS-PAGE and protein concentration was measured spectrophotometrically (Implen Nanophotometer N60, Implen).

For negative stain, purified S protein (0.05 mg/ml) was mixed with VH ab8 (0.02 mg/mL), or soluble ACE2 (0.05 mg/mL) and incubated on ice for 15 mins. For the competition experiment, the S protein (0.05 mg/ml) was first incubated on ice with VH ab8 at concentrations of 0.02, 0.05, 1.00 mg/ml for 30 mins, followed by addition of ACE2 (0.05 mg/mL) for another 30 mins. Grids 

For cryo-EM, grids were plasma cleaned using an H2/O2 gas mixture for 15 seconds in a Solarus II Plasma Cleaner (Gatan Inc.), before 1.8 µL of protein suspension was applied to the surface of the grid. Using a Vitrobot Mark IV (Thermo Fisher Scientific), the sample was applied to either Quantifoil Holey Carbon R1.2/1.3 300 mesh grids or UltrAuFoil Holey Gold 300 mesh grids at a temperature of 10 ºC and a humidity level of 100% and then vitrified in liquid ethane after blotting for 12 seconds with a blot force of -10. All cryo-EM grids were screened using a 200 kV Glacios transmission electron microscope (ThermoFisher Scientific) equipped with a Falcon4 direct electron detector followed by high-resolution data collection on a 300 kV Titan Krios G4 transmission electron microscope (ThermoFisher Scientific) equipped with a Falcon4 direct electron detector in electron event registration (EER) mode. Movies were collected at 155,000x magnification (physical pixel size 0.5 Å) over a defocus range of -1 µm to -3 µm with a total dose of 40 e -/ Å 2 using EPU automated acquisition software (ThermoFisher).

In general, all data processing was performed in cryoSPARC v. After extraction, particles were imported into cryoSPARC and subjected to 2D classification and 3D heterogeneous classification. Final density maps were obtained by 3D homogeneous refinement. For cryo-EM data, motion correction in patch mode, CTF estimation in patch mode, reference-free particle picking and particle extraction were performed on-the-fly in cryoSPARC.

After preprocessing, particles were subjected to 2D classification and 3D heterogeneous classification. The consensus maps were obtained by 3D homogeneous refinement with per particle CTF estimation and aberration correction. Local refinements with the mask covering single RBD and its bound VH ab8 resulted in improvement of the RBD-Ab8 interface. Overall resolution and locally refined resolutions were according to the gold-standard FSC (23) .

Coordinates of PDB 7CH5_H were used as initial models to build the VH ab8 structure.

Individual domains of SARS-CoV-2 HexaPro S trimer (PDB code 6XKL) were docked into cryo-EM density using UCSF Chimera v.1.15 (24) . Initial models were first refined against sharpened locally refined maps, followed by iterative rounds of refinement against consensus map in COOT v.0.9.3 (25) and Phenix v.1.7.1 (26) . Glycans were added at N-linked glycosylation sites in COOT. Model validation was performed using MolProbity (27 SARS-CoV-2 S and SARS_CoV-2 S N501Y pseudotyped retroviral particles were produced in HEK293T cells as described previously (29) . Briefly, a 3rd generation lentiviral packaging system was utilized in combination with plasmids encoding the full-length SARS-CoV-2 spike, along with a transfer plasmid encoding luciferase and GFP as a dual reporter gene.

Pseudoviruses were harvested 60 hrs after transfection, filtered with 0.45 µm PES filters, and frozen. For cell-entry and neutralization assays, HEK293T-ACE2 cells were seeded in 96 well plates at 50,000 cells per well. The next day, pseudoviral preparations normalized for p24 levels (Lenti-X™ GoStix™ Plus) were incubated with dilutions of the indicated antibodies, ACE2-mFc (SinoBiological), or media alone for 1 hr at 37°C prior to addition to cells and incubation for 48 hrs. Cells were then lysed and luciferase activity assessed using the ONE-Glo™ EX Luciferase Assay System (Promega) according to the manufacturer's specifications. Detection of relative luciferase units was carried out using a Varioskan Lux plate reader (ThermoFisher). Percent neutralization was calculated relative to signals obtained in the presence of virus alone for each experiment.

Enzyme-Linked Immunosorbent Assay (ELISA) 100 µl of wild type or N501Y SARS-CoV-2 S protein preparations were coated onto 96-well MaxiSorp™ plates at 2 µg/ml in PBS overnight at 4°C. All washing steps were performed 5 times with PBS + 0.05% Tween 20 (PBS-T). After washing, wells were incubated with blocking buffer (PBS-T + 2% BSA) for 1 hr at room temperature. After washing, wells were incubated with dilutions of VH -FC ab8 or ACE2-mFC (SinoBiological) in PBS-T + 0.5% BSA buffer for 1 hr at room temperature. After washing, wells were incubated with either goat antihuman IgG (Jackson ImmunoResearch) or goat anti-Mouse IgG Fc Secondary Antibody, HRP (Invitrogen) at a 1:8,000 dilution in PBS-T + 0.5% BSA buffer for 1 hr at room temperature.

After washing, the substrate solution (Pierce™ 1-Step™) was used for color development according to the manufacturer's specifications. Optical density at 450 nm was read on a Varioskan Lux plate reader (Thermo Fisher Scientific).For ACE-2 competition assays, experiments were conducted as described above with amendments. Serial dilutions of VH -FC ab8 were incubated for 30 mins at room temperature prior to the addition of 2.5 nM ACE2-mFC (SinoBiological). Wells were then further incubated for 45 minutes at room temperature. After washing, wells were incubated at a 1:8,000 dilution of Goat anti-Mouse IgG Fc Secondary Antibody, HRP (Invitrogen) in PBS-T + 0.5% BSA buffer for 1 hr at room temperature. After 

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