Fig 1.
SR31 binds RBD with high affinity but does not perturb ACE2 binding.
(A) FSEC of RBD in the absence (black) and presence (blue) of SR31. (B) Bio-layer interferometry (BLI) assay with RBD immobilized and SR31 as analyte at three concentrations (nM). (C) Analytic fluorescence-activated cell sorting (FACS) of HEK293T cells expressing the full-length S without staining (red), or with staining in the presence of PBS (i) or indicated sybodies (ii-iv) (cyan). (D) Pull-down of S expressed on the surface of SARS-CoV-2 pseudoviruses using His-tagged sybodies immobilized on Ni-NTA resin. Lanes 1–4 show the elution and lanes 5–8 show the input. Immunoblot bands of the full-length S and the S1 subunit were detected using anti-S1 antibody and conjugated secondary antibodies. Characteristically [4], the full-length S of SARS-CoV-2 is mostly processed. Images of the blot with different exposure are shown (i, ii). The bright-field image of the prestained molecular marker was merged with the chemiluminescent image of the immunoblots. (E) SR31 does not inhibit ACE2 binding. An RBD-coated sensor saturated with SR31 was soaked in 50 nM of SR31 with (blue) and without (black) 25 nM ACE2. As a control, the assay was performed with RBD immobilized and ACE2 as analyte (red). (F) Purification (SEC and SDS-PAGE) and crystallization of the RBD-SR31 complex. Void volume (Vo) and total volume (Vt) are appropriately labeled.
Table 1.
Data collection and refinement statistics.
Fig 2.
Crystal structure of the SR31-RBD complex.
(A) The overall structure of SR31 (light blue) in complex with RBD (grey) which contains Asn343-linked glycans (cyan). The expanded view highlights a deep hydrophobic pocket (green) for CDR3 binding. (B) The overall structure viewed at a different angle. (C) 2Fo-Fc map of the Asn343-linked glycans. MAN, mannose; BMA, β-D-mannose; FUC, fucose; NAG, N-acetylglucosamine. (D-G) Detailed interactions between RBD and the CDR1 (D), CDR2 (E), and CDR3 (F, G). The hydrophobic network formed between CDR3 (orange) and the hydrophobic pocket in RBD (grey) is shown in G. Residues from SR31 are labeled with black texts and residues from RBD are labeled with grey texts. Dash lines indicate hydrogen bonds or salt bridges within 3.6 Å.
Fig 3.
SR31 engages RBD at a site distal to the receptor-binding motif.
(A) Comparison of the SR31 epitope with epitopes for other RBD-targeting nanobodies [22, 35, 36, 39] and mAbs [13–15, 19, 20, 23, 24, 26–28]. Red, the collective epitope of RBM-binders; blue, the SR31 epitope; magenta, the collective epitope of CR3022 and EY6A; green, the COVA1-16 epitope; cyan, the overlap between the epitopes of the RBM binders and COVA1-16; orange, the overlap between the CR3022/EY6A/COVA1-16 and SR31 epitope. (B) SR31 (blue) binds to RBD (grey) at a surface distal to the binding site of ACE2 (red). (C) Comparison of the binding mode between SR31 (blue) and three mAbs including CR3022 (orange and wheat), EY6A (green and pale green), and COVA1-16 (pink and magenta). RBD is shown as white surface with RBM highlighted in red. (D) The binding site of SR31 in the context of the S trimer at its pre-fusion ‘open’ state with one RBD in the ‘up’ conformation and two in the ‘down’ conformation. The structure (PDB ID 6yvb) [3] is viewed from the ‘top’ (perpendicular to the viral membrane). The SR31 epitope is shown in blue. The three RBDs are colored green. SR31 (magenta cartoon) is aligned to the S trimer (surface presentation) by superposing the SR31-RBD structure to each of the RBD. ‘+’, no or minor clashes; ‘-’, with severe clashes.
Fig 4.
SR31 causes dramatic structural arrangements of RBD at the binding site without distorting the receptor-binding motif.
(A, B) The overview (A) and expanded view (B) of the comparison between the ACE2-bound RBD (grey) and SR31-bound RBD (blue). SR31-binding deforms the RBD at the binding site (green) but not at the RBM region (yellow circle). The two SR31 CDRs involved in the deformation are colored wheat. In B, two structural rearrangements (green) are shown at a different angle. The α383–388 helix in the ACE2-bound form is pushed towards the RBD core, and the short helix α364–370 is transformed into a β-strand (β367–370) which forms a parallel β-sheet with β102–104 from SR31 CDR3. (C) An indirect stability assay of the RBD using fluorescence-detection size exclusion chromatography. The RBD was incubated at 4°C (black), 90°C (red), and 99°C (blue) for 20 min before loaded onto an analytical column for gel filtration. The retention profile of RBD was monitored by intrinsic tryptophan fluorescence. The void volume is 1.9 mL and the total volume is 4.5 mL. The chromatogram beyond 3.5 mL (not displayed) only showed background-level fluorescence. (D) Three disulfide bonds (orange spheres) segregate the two motifs (α383–388 and β367–370, green) from the RBM (orange cycle). α383–388 is tethered between Cys379/432 and Cys391/525; β367–360 is tethered between Cys379/432 and Cys336/361.
Fig 5.
SR31 could pair with RBM nanobodies to bind RBD.
(A, B) SR31 does not interfere with MR17 (A) or MR6 (B) for RBD-binding. In A, an RBD-coated sensor was pre-saturated in 200 nM of SR31 before incubating with SR31 alone (black) or a mixture (blue) of SR31 and MR17/MR6. In B, the sensor was saturated with MR6 before analyzed with SR31. For control purposes, the binding between RBD and the sybody used in the pre-incubation was also characterized (red). (C) Alignment of the biparatopic sybody (MR17-SR31)-RBD structure (MR17, grey; SR31, light blue; RBD, red) with the MR17-RBD structure (RBD, green) (PDB ID 7c8w) [35].
Fig 6.
SR31 increases binding affinity and neutralization activity of two fusion partners.
(A) BLI binding assay with immobilized RBD and the biparatopic sybody MR17-SR31 as analyte at increasing concentrations (nM). (B) Neutralization assay of MR17 (blue), MR17-MR17 (magenta), MR17-SR31 (black), the equimolar mix of MR17 and SR31 (MR17+SR31, green), and SR31-SR31 (red). (C, D) Binding kinetics for the RBD-binding by MR6 (C) or by MR6-SR31 (D). (E) Neutralization assay of MR6 (black), MR6-MR6 (red), MR6-SR31 (blue), and the equimolar mix of MR6 and SR31 (MR6+SR31, green). (F, G, H) BLI binding assay with immobilized RBD and the monoparatopic divalent sybody SR31-SR31 (F), MR17-MR17 (G), or MR6-MR6 (H) as analyte at increasing concentrations (nM). (I) Summary of the comparison between monovalent sybodies, SR31-fusion (biparatopic) sybodies, homo-fusion (monoparatopic) sybodies for binding kinetics and neutralization activities. Binding kinetics and neutralization data for MR17 and MR3 are from reference [35]. N.D., not determined; N.A., not applicable. In B and E, data are mean ± standard deviation from three independent experiments, and the x-axis indicates the concentration of the individual sybodies as opposed to the total sybody concentration.