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. 2020 Sep 29;33(3):108274. doi: 10.1016/j.celrep.2020.108274

An Alternative Binding Mode of IGHV3-53 Antibodies to the SARS-CoV-2 Receptor Binding Domain

Nicholas C Wu 1,2,9, Meng Yuan 3,9, Hejun Liu 3,9, Chang-Chun D Lee 3, Xueyong Zhu 3, Sandhya Bangaru 3, Jonathan L Torres 3, Tom G Caniels 4, Philip JM Brouwer 4, Marit J van Gils 4, Rogier W Sanders 4,5, Andrew B Ward 3,6,7, Ian A Wilson 3,6,7,8,10,∗
PMCID: PMC7522650  PMID: 33027617

Summary

IGHV3-53-encoded neutralizing antibodies are commonly elicited during SARS-CoV-2 infection and target the receptor-binding domain (RBD) of the spike (S) protein. Such IGHV3-53 antibodies generally have a short CDR H3 because of structural constraints in binding the RBD (mode A). However, a small subset of IGHV3-53 antibodies to the RBD contain a longer CDR H3. Crystal structures of two IGHV3-53 neutralizing antibodies here demonstrate that a longer CDR H3 can be accommodated in a different binding mode (mode B). These two classes of IGHV3-53 antibodies both target the ACE2 receptor binding site, but with very different angles of approach and molecular interactions. Overall, these findings emphasize the versatility of IGHV3-53 in this common antibody response to SARS-CoV-2, where conserved IGHV3-53 germline-encoded features can be combined with very different CDR H3 lengths and light chains for SARS-CoV-2 RBD recognition and virus neutralization.

Keywords: COVID-19, SARS-CoV-2, antibodies, x-ray crystallography, spike protein, receptor-binding domain

Graphical Abstract

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Highlights

  • •

    Crystal structures of IGHV3-53 antibodies that frequently bind SARS-CoV-2 RBD

  • •

    Binding modes (A and B) of these IGHV3-53 antibodies depend on CDR H3 length

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    Germline-encoded CDR H1 and H2 motifs dominate the two binding poses

  • •

    CDR H3 length of IGHV3-53 antibodies is associated with light chain preference

Antibodies to the SARS-CoV-2 receptor-binding domain are commonly encoded by IGHV3-53, and most have a short CDR H3. Wu et al. show that IGHV3-53 antibodies with a long CDR H3 adopt an alternative binding mode, demonstrating that IGHV3-53 is even more versatile than previously thought in targeting SARS-CoV-2.

Introduction

Development of an effective vaccine against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is perhaps the most exigent health-related priority because of the ongoing coronavirus disease 2019 (COVID-19) pandemic. The molecular and functional understanding of the antibody response to SARS-CoV-2 infection and vaccination is critical for vaccine assessment and redesign. Most SARS-CoV-2 antibodies that target the receptor-binding domain (RBD) on the spike (S) protein appear to be neutralizing (Brouwer et al., 2020; Cao et al., 2020; Robbiani et al., 2020; Rogers et al., 2020; Zost et al., 2020), and the most intuitive mechanism of neutralization is that they block binding of the host receptor angiotensin-converting enzyme 2 (ACE2).

To date, several structures of antibodies that target the ACE2-binding site on RBD have been determined (Cao et al., 2020; Ju et al., 2020; Shi et al., 2020), including some that are encoded by the IGHV3-53 gene (Barnes et al., 2020b; Brouwer et al., 2020; Wu et al., 2020; Yuan et al., 2020a). Our previous study demonstrated that antibodies encoded by the IGHV3-53 gene use germline-encoded residues to engage the ACE2-binding site on the RBD, accounting for their frequency in shared antibody responses in SARS-CoV-2 patients (Yuan et al., 2020a). Because of structural constraints in their mode of binding through interaction with the germline-encoded heavy chain complementarity determining regions (CDRs) H1 and H2, a short CDR H3 (length ≤ 10 amino acids, Kabat numbering) is also a molecular signature of these IGHV3-53 antibodies (Barnes et al., 2020b; Yuan et al., 2020a). Nevertheless, a small subset (about 10%) of RBD-targeting IGHV3-53 antibodies have much longer CDR H3s (15 amino acids or longer) (Barnes et al., 2020b; Yuan et al., 2020a). As it was not apparent how such IGHV3-53 antibodies could retain the same binding mode and fit their longer CDR H3 into a restricted space between the antibody and the RBD (Yuan et al., 2020a), we aimed to resolve this conundrum.

Results

Two RBD-Targeting IGHV3-53 Antibodies with Different Binding Modes

We determined crystal structures of two IGHV3-53 neutralizing antibodies, COVA2-04 and COVA2-39 (Brouwer et al., 2020), with different CDR H3 lengths in complex with SARS-CoV-2 RBD to 2.35 and 1.72 Å resolution, respectively (Figure 1A; Table S1). Both antibodies were derived from a convalescent donor from Amsterdam and potently neutralize SARS-CoV-2 virus (Brouwer et al., 2020). Similar to typical RBD-targeting IGHV3-53 antibodies (Barnes et al., 2020b; Wu et al., 2020; Yuan et al., 2020a), COVA2-04 has a relatively short CDR H3 of 10 amino acids, whereas COVA2-39 CDR H3 is 15 amino acids (Kabat numbering; Figure S1A). COVA2-04 has only two somatic amino acid substitutions in the heavy chain and one in the light chain, which is encoded by IGKV3-20 (Figure S1B). COVA2-39 has three somatic mutations in the heavy chain and one in the light chain, which is encoded by IGLV2-23 (Figure S1C).

Figure 1.

Figure 1

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Structures of Two IGHV3-53 Antibodies to SARS-CoV-2 RBD with Very Different Binding Modes

(A) Crystal structures of COVA2-04/RBD and COVA2-39/RBD complexes are shown. Human ACE2/RBD complex is also shown for comparison (PDB: 6M0J) (Lan et al., 2020).

(B) Zoomed-in views of COVA2-04/RBD (left) and COVA2-39/RBD (right) interfaces are shown. COVA2-04 (cyan) and COVA2-39 (pink) are shown in surface representation and RBD (white) in a cartoon representation in the same view as (A). The ACE2-binding ridge in the RBD (residues 471–491) is in orange.

(C) Binding modes of COVA2-04 (cyan), COVA2-39 (pink), CC12.1 (green), CC12.3 (orange), B38 (gray), and CV30 (salmon) to SARS-CoV-2 (white) are compared in the same view as in (A) and (B). CC12.1/RBD, CC12.3/RBD, RBD B38/RBD, and CV30/RBD complexes are from PDB: 6XC3 and PDB: 6XC4 (Yuan et al., 2020a), PDB: 7BZ5 (Wu et al., 2020), and PDB: 6XE1 (Hurlburt et al., 2020), respectively. The N-glycan observed at SARS-CoV-2 RBD N343, which is distant from the epitopes of COVA2-04 and COVA2-39, is shown in red.

(D) Binding kinetics of COVA2-04 and COVA2-39 Fabs against SARS-CoV-2 RBD were measured using biolayer interferometry (BLI). The y axis represents the response. Blue lines represent the response curves and red lines represent a 1:1 binding model. Binding kinetics were measured for four concentrations of each Fab at 2-fold dilution starting from 125 nM. The Kd (mean ± SD) and R2 of the fitting are indicated. Representative results of two replicates are shown here.

COVA2-04 and COVA2-39 both bind to the ACE2-binding site on RBD, which is consistent with previous competition assays (Brouwer et al., 2020). Nonetheless, their angles of approach and binding modes are very different (Figures 1A–1C). COVA2-04 mainly uses the light chain to interact with the flat surface of the ACE2-binding site and the heavy chain with the RBD ridge, whereas COVA2-39 mainly uses the heavy chain to interact with the flat surface and both heavy and light chains with the ridge (Figures 1B, 2A, and 2B; Table S2). In addition, COVA2-04 binds to the side of the ridge, whereas COVA2-39 binds at its tip (Figure 1B). The binding mode of COVA2-04 is very similar to previously characterized IGHV3-53 antibodies with a short CDR H3, including CC12.1, CC12.3, B38, CV30, and C105 (Barnes et al., 2020b; Hurlburt et al., 2020; Wu et al., 2020; Yuan et al., 2020a) (binding mode A; Figure 1C). In contrast, binding mode (mode B) of COVA2-39 is quite different, and its Fab is rotated 180° along its long axis relative to COVA2-04, thereby swapping the relative orientation of the light and heavy chains, resulting in completely different molecular interactions.

Figure 2.

Figure 2

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Epitopes of COVA2-04 and COVA2-39

(A and B) Epitope residues of (A) COVA2-04 and (B) COVA2-39 are identified by PISA (Krissinel and Henrick, 2007). Epitope residues contacting the heavy chain are in orange and light chain in yellow. In the left panels, CDR loops are labeled. In the right panels, epitope residues are labeled. For clarity, only representative epitope residues are labeled. Epitope residues that are also involved in ACE2 binding are in red. The location of ACE2-binding ridge in the RBD (residues 471–491) is indicated by the black outline.

(C) ACE2-binding residues on the RBD are shown in blue. In the left panel, ACE2 is shown in green within a semi-transparent ribbon representation. In the right panel, ACE2-binding residues are labeled. A total of 17 residues in the SARS-CoV-2 RBD are used for binding by ACE2 (Lan et al., 2020). The 17 ACE2-binding residues are as described previously (Lan et al., 2020).

Both Binding Modes Are Dominated by Heavy Chain

Structural modeling shows that both COVA2-04 and COVA2-39 can bind to the RBD only when it is in the “up” conformation on the trimeric S protein (Figure S2) (Walls et al., 2020; Wrapp et al., 2020). This finding is consistent with previous low-resolution, negative-stain electron microscopy analysis, which also indicated that these antibodies have different angles of approach to the spike protein, but both bind to the RBD one-up conformation (Brouwer et al., 2020). Despite these differences, interactions of COVA2-04 and COVA2-39 with the RBD are both dominated by the heavy chain. For COVA2-04, the buried surface areas (BSA) of the heavy and light chains are 798 and 360 Å2, respectively, compared with 576 and 128 Å2 for COVA2-39. However, this BSA difference does not translate into a corresponding difference in Fab binding affinity. Specifically, the dissociation constants (Kd) for COVA2-04 and COVA2-39 to insect cell-expressed RBD are 40 and 21 nM, respectively (Figure 1D). COVA2-04 exhibits slow-on/slow-off kinetics, whereas COVA2-39 has fast-on/fast-off kinetics. Despite the faster off-rate, similar Kd, and lower BSA, COVA2-39 IgG is more potent than COVA2-04 in neutralizing SARS-CoV-2 (half maximal inhibitory concentration [IC50] values of 0.036 and 0.22 μg/mL, respectively, in a pseudovirus assay, and 0.054 and 2.5 μg/mL in an authentic virus assay (Brouwer et al., 2020)).

The COVA2-39 epitope has less overlap with the ACE2-binding site than does the epitope of COVA2-04. Among 17 ACE2-binding residues on the RBD (Lan et al., 2020), 16 are within the epitope of COVA2-04 and 11 within the epitope of COVA2-39 (Figures 2A–2C). The difference in angles of approach and apparent avidity of the IgG interaction in the context of the spike trimer appear to allow COVA2-39 to attain higher neutralization potency, similar to avidity effects for IgG of some antibodies to influenza hemagglutinin RBD (Ekiert et al., 2012; Lee et al., 2014). Indeed, the Kd of IgG to mammalian cell-expressed SARS-CoV-2 spike protein is 2.3 nM for COVA2-04 and 0.1 nM for COVA2-39 (Brouwer et al., 2020), which represents a 20-fold difference in apparent IgG binding avidity that may also result from the higher local Fab concentration and rebinding of IgG to the spike protein on both the sensor in the binding experiment and on the viral surface. Such a difference in binding avidity effect between COVA2-04 and COVA2-39 may be attributed to their differential binding modes. Although both COVA2-04 and COVA2-39 bind to the RBD in the one-up conformation (Brouwer et al., 2020), because of steric hindrance with the adjacent RBD, such steric hindrance seems to be more extensive for COVA2-04 than COVA2-39 (Figures S2C–S2D). As a result, COVA2-39 may be able to bind to the RBD in more configurations of the RBD on the spike than COVA2-04 and increase the effect of IgG binding avidity. However, this explanation is a hypothesis and requires further experimental evidence to validate.

Both Binding Modes Involve Similar Motifs

Previously, we have described the germline-encoded features of IGHV3-53, including an 32NY33 motif in CDR H1 and an 53SGGS56 motif in CDR H2, which facilitate interaction with the ACE2-binding site of SARS-CoV-2 RBD in binding mode A (Yuan et al., 2020a). These motifs are also important for COVA2-04 engagement of the RBD in a similar manner to other IGHV3-53 antibodies in binding mode A (Figures 3A and 3B) (Yuan et al., 2020a). Interestingly, some of these germline-encoded residues are also involved in binding of COVA2-39 (mode B), but to a different location and, hence, to different residues on the RBD. VH Y33 of the 32NY33 motif is retained and forms a π-π stacking interaction between its aromatic ring with the aromatic side chain of Y489 (Figure 3C). Although VH N32 in COVA2-39 does not interact with the RBD, both its side chain and main chain participate in a 310 turn to stabilize the CDR H1 backbone (Figure 3C), as observed with COVA2-04 and other IGHV3-53 antibodies (Figure 3A) (Yuan et al., 2020a). The 53SGGS56 is somatically mutated to 53TGGT56 in COVA2-39, which would appear to be a conservative substitution. Similar to the 53SGGS56 motif in binding mode A (Figure 3B), the 53TGGT56 motif in COVA2-39 also forms an extensive hydrogen-bond (H-bond) network (Figure 3D), but to a different region of SARS-CoV-2 RBD (Figure S3). The 53TGGT56 motif in COVA2-39 extensively H-bonds with RBD E484 through the side chains of VH T53 and VH T56 (water-mediated H-bond), as well as the backbone amides of VH T53, VH G55, and VH T56 (Figure 3D). The side chains of VH T53 and VH T56 also participate in additional water-mediated H-bonds with backbone carbonyls and amides of the RBD. Nevertheless, despite the similarity between Ser and Thr in size and ability to form similar H-bonds with their side-chain hydroxyl, reverting the 53TGGT56 motif in COVA2-39 to the germline-encoded 53SGGS56 motif decreased its Kd to SARS-CoV-2 RBD by at least 50-fold (Figure 1D; Figure S4A). The increased binding for 53TGGT56 is likely due to the methyl groups in VH T53 and T56, which make additional van der Waals interactions (Figure S4B).

Figure 3.

Figure 3

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Heavy Chain Interactions of COVA2-39 and COVA2-04 with the RBD

(A and C) Interactions are shown between RBD (white) and signature 32NY33 motifs on the CDR H1 loop of VH3-53 antibodies (A) COVA2-04 (cyan) and (C) COVA2-39 (pink).

(B and D) RBD forms an extensive hydrogen bonding network with (B) 53SGGS56 motif on the CDR H2 loop of COVA2-04, and (D) 53TGGT56 motif on the CDR H2 loop of COVA2-39.

(E) A π-π interaction is illustrated between G485 peptide backbone in the RBD (semi-transparent white surface) and VH Y52. Hydrogen bonds are represented by dashed lines and water molecules by red spheres.

Like the 53SGGS56 motif, 53TGGT56 in COVA2-39 takes part in a type I β-turn along with VH Y52, which is the first residue (i) in the turn (YTGG). The partial positive dipole from the aligned, up-pointing amides on one face of the β-turn (Sheridan and Allen, 1980; Zhang et al., 2015) forms an electrostatic interaction with the side-chain carboxyl of RBD E484. In addition, VH R71 hydrogen bonds to the 53TGGT56 motif and may have a role in stabilizing the interaction between the 53TGGT56 motif and RBD E484 (Figure 3D). VH R71 also forms a partial dipole interaction with the other face of the β-turn, where E484, 53TGGT56 β-turn, and VH R71 are aligned. Moreover, VH Y52 is an important residue for binding SARS-CoV-2 RBD in binding mode B, but not in binding mode A (Figure 3E). VH Y52 of COVA2-39 forms a π-π interaction with backbone peptide bond between RBD G485 and F486. COVA2-39 also uses other IGHV3-53 germline-encoded residues for interaction with the RBD, including VH S30, VH Y58, VH N73, and VH S74 (Figures S4C and S4D). Overall, these findings demonstrate that the germline-encoded features of IGHV3-53 are conducive for interaction with different regions of the ACE2-binding site on the RBD that involve different approach angles (i.e., binding modes A and B) in the context of different CDR H3 lengths.

Binding Mode B Has Fewer Structural Constraints on CDR H3 Length

Next we aimed to understand the relationship between CDR H3 length and the two different binding modes. Consistent with previous structures of IGHV3-53 antibodies that target the RBD in binding mode A (Barnes et al., 2020b; Wu et al., 2020; Yuan et al., 2020a), CDR H3 of COVA2-04 is highly buried by the RBD and the light chain (Figure 4A). This observation substantiates the notion that IGHV3-53 in binding mode A has strong structural constraints on CDR H3 length. In contrast, in binding mode B, the longer CDR H3 of COVA2-39 is largely solvent exposed (Figure 4B). This observation explains why IGHV3-53 can accommodate a longer CDR H3 in binding mode B (COVA2-39) than in mode A (COVA2-04, CC12.1, CC12.3, B38, CV30, and C105). COVA2-39 CDR H3 interacts with the RBD mostly through non-specific van der Waals interactions. Three H-bonds are also made, two of which (one water-mediated) involve the CDR H3 backbone, whereas only one involves a side chain (VH E100c) (Figure 4C). Thus, interaction between CDR H3 of COVA2-39 and RBD is largely sequence non-specific and appears to simply accommodate its longer length, although one side chain out of 15 CDR H3 residues (VH F100g) appears to be play a role in stabilizing the interaction between light chain and RBD (see below; Figures 5A and 5B). The CDR H3 sequences of IGHV3-53 antibodies in binding mode A (COVA2-04, CC12.1, CC12.3, B38, CV30, and C105) are also quite different (Wu et al., 2020; Yuan et al., 2020a) (Figure 4D). As a result, although both binding modes A and B do not have strong constraints on the actual CDR H3 sequences, structural constraints on CDR H3 length is much stronger in binding mode A than in mode B.

Figure 4.

Figure 4

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Structural Constraints of CDR H3 Length in Different Binding Modes of IGHV3-53 Antibodies

(A) Interaction between COVA2-04 (cyan) and the RBD (white) is shown with CDR H3 highlighted in red.

(B) Interaction between COVA2-39 (pink) and the RBD (white) is shown with CDR H3 highlighted in red. In this view, the RBD is rotated ~180° relative to (A).

(C) CDR H3 of COVA2-39 makes very few contacts with the RBD. Hydrogen bond interactions are represented by dashed lines and water molecules by red spheres.

(D) CDR H3 sequences are compared for RBD-targeting IGHV3-53 antibodies with short a CDR H3, including COVA2-04, CC12.1 (Yuan et al., 2020a), CC12.3 (Yuan et al., 2020a), B38 (Wu et al., 2020), C105 (Barnes et al., 2020b), CV30 (Hurlburt et al., 2020), COVA2-07 (Brouwer et al., 2020), and BD-494 (Cao et al., 2020). Of note, BD-494 belongs to a cluster of RBD-targeting IGHV3-53 antibodies with similar CDR H3 sequences, including BD500, BD-503, BD-505, BD-506, BD-507, and BD-508 (Cao et al., 2020). Residue positions are labeled according to the Kabat numbering scheme.

Figure 5.

Figure 5

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Structural Analysis of the RBD Ridge-Anchoring Pocket in COVA2-39

(A) The RBD is shown as white cartoon. The region of the ACE2-binding ridge (residues 471–491) is highlighted in orange. F486 at the tip of the ridge is shown as a stick representation and COVA2-39 Fab in a surface representation.

(B) Interaction between F486 and COVA2-39 in the ridge-anchoring pocket is shown.

(C) CDR H3 length of previously discovered RBD-targeting IGHV3-53 antibodies is summarized by box-and-whisker plots (Brouwer et al., 2020; Cao et al., 2020; Ju et al., 2020; Rogers et al., 2020; Wu et al., 2020). The middle line represents the median. The lower and upper hinges of the box plot correspond to the first and third quartiles (the 25th and 75th percentiles). The upper whisker extends from the upper hinge to the largest value no further than 1.5 times inter-quartile range (IQR; the distance between the first and third quartiles) from the upper hinge. The lower whisker extends from the lower hinge to the smallest value at most 1.5 times IQR of the lower hinge. The light chain genes for these antibodies are shown on the x axis.

Light Chain of IGHV3-53 Antibody Is Associated with CDR H3 Length

An important feature in COVA2-39 interaction with the RBD is its engagement of the ACE2-binding ridge. Specifically, RBD F486 at the tip of the ridge is anchored in a pocket formed by both heavy and light chains (Figure 5A). RBD F486 forms a π-π stacking interaction with VL Y91, which in turn is stabilized by a network of T-shaped π-π stacking interactions involving VH W47, VH F100g, VL Y30, and VL W96 (Figure 5B; Figure S5). This critical interaction with the ridge indicates that the light-chain identity is important for binding mode B. In fact, the light chain use of RBD-targeting IGHV3-53 antibodies segregates between those with short CDR H3s and those with long CDRs, although some may be able to pair with both (Figure 5C) (Barnes et al., 2020a; Yuan et al., 2020a). For example, RBD-targeting IGHV3-53 antibodies that have a relatively short CDR H3 (7–11 amino acids, n = 62 in our analysis) predominantly pair with IGKV1-9 (n = 20) and IGKV3-20 (n = 15) (Figure 5C) (Brouwer et al., 2020; Cao et al., 2020; Ju et al., 2020; Robbiani et al., 2020; Rogers et al., 2020; Wu et al., 2020). In contrast, IGKV1-9 and IGKV3-20 are not used to date in RBD-targeting IGHV3-53 antibodies that have a long CDR H3 (15 amino acids or longer, n = 12), which predominantly pair with IGLV2-14 (n = 6) (Figure 5C) (Barnes et al., 2020a; Yuan et al., 2020a). Therefore, despite the limited sample size, there seems to be a relationship between the light-chain identity of RBD-targeting IGHV3-53 antibodies and CDR H3 length. This observation further supports a role for light-chain identity, together with CDR H3 length, in determining the particular binding mode of IGHV3-53 antibodies to SARS-CoV-2 RBD.

Discussion

Why is binding mode A seen more commonly than mode B, given the structural constraints for below-average CDR H3 length, even for VH3-53 antibodies in general (Barnes et al., 2020b; Wu et al., 2020; Yuan et al., 2020a)? In mode A, the germline sequences of CDR H1 and H2, along with a conserved residue in FR3, seems to be the major determinants of binding without much involvement of specific residues of CDR H3. In binding mode B, formation of the RBD ridge-anchoring pocket involves both CDR H3 and L3 loops. Thus, the probability of satisfying the molecular requirements to create the RBD ridge-anchoring pocket (binding mode B) could be lower than simply using the unmutated germline sequence with no particular CDR H3 sequence requirements other than length (binding mode A). Very recently, an IGHV3-53 antibody C144, which has a CDR H3 length of 23, was shown to bind to the RBD with similarity to COVA2-39, especially around the 53SGGS56 motif (Barnes et al., 2020a). Specifically, the 53SGGS56 motif in C144 forms an extensive H-bond network with RBD E484 (Barnes et al., 2020a), similar to how COVA2-39 H-bonds with RBD E484 using its 53TGGT56 motif. This observation substantiates the notion that RBD-targeting IGHV3-53 antibodies with a long CDR H3 tend to adopt alternative binding mode B.

Despite the large number of neutralizing antibodies currently being identified (Andreano et al., 2020; Brouwer et al., 2020; Cao et al., 2020; Chi et al., 2020; Ju et al., 2020; Kreer et al., 2020; Robbiani et al., 2020; Rogers et al., 2020; Seydoux et al., 2020; Zost et al., 2020), structural understanding of the antigenicity and immunogenicity of SARS-CoV-2 S protein is still at an early stage. Nevertheless, it is clear that potent antibodies can be elicited to the RBD in natural infection. As SARS-CoV-2 may eventually become endemic within the human population (Li et al., 2020) and escape mutations may arise, the structural information elucidated here can be harnessed for modifying or improving existing vaccine designs and for assessing the quality and efficacy of vaccine responses.

STAR★Methods

Key Resources Table

REAGENT or RESOURCE SOURCE IDENTIFIER
ExpiCHO Expression System Kit Thermo Fisher Scientific A29133
Expi293 Expression System Kit ThermoFisher Cat# A14635
HyClone insect cell culture medium GE Healthcare SH30280.03
Phosphate-buffered saline (PBS) Thermo Fisher Scientific 14040133
Ni-NTA Superflow QIAGEN 30450
DH10Bac competent cells Thermo Fisher Scientific 10361012
CaptureSelect CH1-XL Affinity Matrix Thermo Fisher Scientific 2943452010
Chemicals and Recombinant Proteins
DpnI New England Biolabs R0176L
Trypsin New England Biolabs P8101S
Fugene 6 Transfection Regent Promega E2691
Sodium chloride (NaCl) Sigma-Aldrich S9888
Tris Base Sigma-Aldrich 11814273001
Concentrated hydrochloric acid (HCl) Sigma-Aldrich H1758
Sodium azide (NaN3) Sigma-Aldrich S2002
Bovine Serum Albumin (BSA) Sigma-Aldrich A9418
Tween 20 Fisher Scientific BP337-500
Chemicals for protein crystallization Hampton Research N/A
Critical Commercial Assays
In-Fusion HD Cloning Kit Takara 639647
KOD Hot Start DNA Polymerase EMD Millipore 71086-3
PCR Clean-Up and Gel Extraction Kit Clontech Laboratories 740609.250
QIAprep Spin Miniprep Kit QIAGEN 27106
NucleoBond Xtra Maxi Clontech Laboratories 740414.100
Deposited Data
X-ray coordinates and structure factors for COVA2-04/RBD This manuscript PDB 7JMO
X-ray coordinates and structure factors for COVA2-39/RBD This manuscript PDB 7JMP
Cell Lines
ExpiCHO cells Thermo Fisher Scientific A29127
Sf9 cells ATCC CRL-1711
High Five cells Thermo Fisher Scientific B85502
Recombinant DNA
phCMV3-COVA2-04 Fab heavy chain (Brouwer et al., 2020) N/A
phCMV3-COVA2-04 Fab light chain (Brouwer et al., 2020) N/A
phCMV3-COVA2-39 Fab heavy chain (Brouwer et al., 2020) N/A
phCMV3- COVA2-39 Fab light chain (Brouwer et al., 2020) N/A
pFastBac-SARS-CoV-2-RBD (Yuan et al., 2020b) N/A
Software and Algorithms
HKL2000 (Otwinowski and Minor, 1997) N/A
Phaser (McCoy et al., 2007) N/A
Coot (Emsley et al., 2010) N/A
Refmac5 (Murshudov et al., 2011) N/A
MolProbity (Chen et al., 2010) N/A
Octet analysis software 9.0 Fortebio https://www.moleculardevices.com
R https://www.r-project.org/ N/A
Other
Fab-CH1 2nd generation (FAB2G) biosensors ForteBio Cat# 18-5019
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Resource Availability

Lead contact

Information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Ian A. Wilson (wilson@scripps.edu).

Materials availability

All unique/stable reagents generated in this study are available from the Lead Contact (I.A.W.) with a completed Materials Transfer Agreement.

Data and code availability

X-ray coordinates and structure factors have been deposited in the RCSB Protein Data Bank with accession code PDB: 7JMO for COVA2-04/RBD and PDB: 7JMP for COVA2-39/RBD.

Experimental Model and Subject Details

Cell cultures

ExpiCHO cells were maintained according to the manufacturer’s instructions (Thermo Fisher Scientific). Sf9 cells (Spodoptera frugiperda ovarian cells, female) and High Five cells (Trichoplusia ni ovarian cells, female) were maintained HyClone insect cell culture medium.

Method Details

Expression and purification of SARS-CoV-2 RBD

The receptor-binding domain (RBD) (residues 319-541) of the SARS-CoV-2 spike (S) protein (GenBank: QHD43416.1) was cloned into a customized pFastBac vector (Ekiert et al., 2011), and fused with an N-terminal gp67 signal peptide and C-terminal His6 tag (Yuan et al., 2020b). A recombinant bacmid DNA was generated using the Bac-to-Bac system (Life Technologies). Baculovirus was generated by transfecting purified bacmid DNA into Sf9 cells using FuGENE HD (Promega), and subsequently used to infect suspension cultures of High Five cells (Life Technologies) at an MOI of 5 to 10. Infected High Five cells were incubated at 28 °C with shaking at 110 rpm for 72 h for protein expression. The supernatant was then concentrated using a 10 kDa MW cutoff Centramate cassette (Pall Corporation). The RBD protein was purified by Ni-NTA, followed by size exclusion chromatography, and buffer exchanged into 20 mM Tris-HCl pH 7.4 and 150 mM NaCl.

Expression and purification of Fabs

For COVA2-04 and COVA2-39, the heavy and light chains were cloned into phCMV3. The plasmids were transiently co-transfected into ExpiCHO cells at a ratio of 2:1 (HC:LC) using ExpiFectamine CHO Reagent (Thermo Fisher Scientific) according to the manufacturer’s instructions. The supernatant was collected at 10 days post-transfection. The Fabs were purified with a CaptureSelect CH1-XL Affinity Matrix (Thermo Fisher Scientific) followed by size exclusion chromatography.

Crystallization and structural determination

COVA2-04/RBD and COVA2-39/RBD complexes were formed by mixing each of the protein components at an equimolar ratio and incubated overnight at 4°C. Each complex was adjusted to 12 mg/ml and screened for crystallization using the 384 conditions of the JCSG Core Suite (QIAGEN) on our custom-designed robotic CrystalMation system (Rigaku) at Scripps Research. Crystallization trials were set-up by the vapor diffusion method in sitting drops containing 0.1 μL of protein and 0.1 μL of reservoir solution. Diffraction-quality crystals were obtained in the following conditions:

COVA2-04/RBD complex (12 mg/mL): 8.5% isopropanol, 10% ethylene glycol, 15% glycerol, 0.085 M HEPES pH 7.5, and 17% polyethylene glycol 4000 at 20°C.

COVA2-39/RBD complex (12 mg/mL): 0.1 M sodium citrate pH 5.6, 20% isopropanol, 10% ethylene glycol, and 20% polyethylene glycol 4000 at 20°C.

All crystals appeared on day 3, harvested on day 7, and were then flash cooled and stored in liquid nitrogen until data collection. Diffraction data were collected at cryogenic temperature (100 K) at Stanford Synchrotron Radiation Lightsource (SSRL) on the new Scripps/Stanford beamline 12-1 with a beam wavelength of 0.97946 Å, and processed with HKL2000 (Otwinowski and Minor, 1997). Structures were solved by molecular replacement using PHASER (McCoy et al., 2007). The models for molecular replacement of RBD and COVA2-04 were from PBD 6XC4 (Yuan et al., 2020a), whereas the model of COVA2-39 was generated by Repertoire Builder (https://sysimm.ifrec.osaka-u.ac.jp/rep_builder/) (Schritt et al., 2019). Iterative model building and refinement were carried out in COOT (Emsley et al., 2010) and PHENIX (Adams et al., 2010), respectively. Epitope and paratope residues, as well as their interactions, were identified by accessing PISA (Proteins, Interfaces, Structures and Assemblies) at the European Bioinformatics Institute (https://www.ebi.ac.uk/pdbe/prot_int/pistart.html) (Krissinel and Henrick, 2007). Epitope and paratope residues were defined as having a buried surface area of larger than 0 Å2 upon binding.

Biolayer interferometry binding assay

Antibody binding and competition assays were performed by biolayer interferometry (BLI) using an Octet Red instrument (FortéBio) as described previously (Wu et al., 2017), with anti-human Fab-CH1 2nd generation (FAB2G) biosensors. There were five steps in the assay: 1) baseline: 60 s with 1x kinetics buffer; 2) loading: 240 s with 50 μg/mL of COVA2-04 Fab or COVA2-39 Fab; 3) baseline: 60 s with 1x kinetics buffer; 4) association: 180 s with serial diluted concentrations of SARS-CoV-2 RBD; and 5) dissociation: 180 s with 1x kinetics buffer. For Kd estimation, a 1:1 binding model was used.

Quantification and Statistical Analysis

Summary statistics in the box and whiskers plots in Figure 5C were generated by “geom_boxplot” function of the ggplot2 package in R (https://www.r-project.org/). Statistical details can be found in the Figure legend.

Acknowledgments

We thank Henry Tien for technical support with the crystallization robot, Jeanne Matteson and Yuanzi Hua for contribution to mammalian cell culture, Wenli Yu for contribution to insect cell culture, and Robyn Stanfield for assistance in data collection. We are grateful to the staff of Stanford Synchrotron Radiation Laboratory (SSRL) Beamline 12-1 for assistance. This work was supported by NIH grant R00 AI139445 (N.C.W.); Bill and Melinda Gates Foundation grants OPP1170236 (A.B.W., I.A.W.), OPP1111923, OPP1132237, and INV-002022 (R.W.S.); and NIH HIV Vaccine Research and Design (HIVRAD) grant P01 AI110657 (R.W.S., A.B.W., I.A.W.). M.J.v.G. is a recipient of an AMC Fellowship, and R.W.S is a recipient of a Vici grant from the Netherlands Organisation for Scientific Research (NWO). Use of the SSRL, SLAC National Accelerator Laboratory is supported by the U.S. Department of Energy (DOE), Office of Science, Office of Basic Energy Sciences under contract DE-AC02-76SF00515. The SSRL Structural Molecular Biology Program is supported by the DOE Office of Biological and Environmental Research and by the NIH National Institute of General Medical Sciences (including grant P41GM103393).

Author Contributions

N.C.W., M.Y., H.L., and I.A.W. conceived and designed the study. N.C.W., M.Y., H.L., and C.-C.D.L. expressed and purified the proteins. T.G.C., P.J.M.B., M.J.v.G., and R.W.S. provided antibody clones and sequences. S.B., J.L.T., and A.B.W. provided the nsEM maps and performed fitting. N.C.W., M.Y., H.L., and X.Z. performed the crystallization and X-ray data collection and determined and refined the X-ray structures. N.C.W., M.Y., H.L., C.-C.D.L., X.Z., and I.A.W. analyzed the data. N.C.W., M.Y., H.L., and I.A.W. wrote the paper. All authors reviewed and/or edited the paper.

Declaration of Interests

Amsterdam UMC previously filed a patent application that included SARS-CoV-2 antibodies COVA2-04 and COVA2-39 (Brouwer et al., 2020).

Published: September 29, 2020

Footnotes

Supplemental Information can be found online at https://doi.org/10.1016/j.celrep.2020.108274.

Supplemental Information

Document S1. Figures S1–S5 and Tables S1 and S2
mmc1.pdf (2MB, pdf)
Document S2. Article plus Supplemental Information
mmc2.pdf (5MB, pdf)

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Document S1. Figures S1–S5 and Tables S1 and S2
mmc1.pdf (2MB, pdf)
Document S2. Article plus Supplemental Information
mmc2.pdf (5MB, pdf)

Data Availability Statement

X-ray coordinates and structure factors have been deposited in the RCSB Protein Data Bank with accession code PDB: 7JMO for COVA2-04/RBD and PDB: 7JMP for COVA2-39/RBD.

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