Structure-Based Design with Tag-Based Purification and In-Process Biotinylation Enable Streamlined Development of SARS-CoV-2 Spike Molecular Probes

Abstract

Biotin-labeled molecular probes, comprising specific regions of the SARS-CoV-2 spike, would be helpful in the isolation and characterization of antibodies targeting this recently emerged pathogen. To develop such probes, we designed constructs incorporating an N-terminal purification tag, a site-specific protease-cleavage site, the probe region of interest, and a C-terminal sequence targeted by biotin ligase. Probe regions included full-length spike ectodomain as well as various subregions, and we also designed mutants to eliminate recognition of the ACE2 receptor. Yields of biotin-labeled probes from transient transfection ranged from ~0.5 mg/L for the complete ectodomain to >5 mg/L for several subregions. Probes were characterized for antigenicity and ACE2 recognition, and the structure of the spike ectodomain probe was determined by cryo-electron microscopy. We also characterized antibody-binding specificities and cell-sorting capabilities of the biotinylated probes. Altogether, structure-based design coupled to efficient purification and biotinylation processes can thus enable streamlined development of SARS-CoV-2 spike-ectodomain probes.

Figure 1.. Strategy for Tag-Based Purification with On-Column Biotinylation

(A) Schematic design of the expression construct of SARS-CoV-2 molecular probes. A single chain human Ig constant domain (scFc) was added at N terminus to facilitate expression and purification. The AVI tag was placed at the C terminus after a 10-amino acid linker for biotinylation. The red arrows in the second and fourth Fc domains showed the “knob-in-hole” mutations to prevent dimerization of the scFc. (B) Biotinylation and HRV3C digestion. Cell culture supernatant from cells transiently transfected with plasmid was loaded onto protein A affinity column. Biotinylation and HRV3C cleavage reactions can be carried out in series or simultaneously, as buffers for both reactions are compatible.

Figure 2.. Design of the SARS-CoV-2 Spike S2P Molecular Probe

(A) Schematic of construction of the SARS-CoV-2 spike probe containing the ectodomain of the spike protein, the GSAS mutations replacing the RRAR furin cleavage site and the K986PV987P (2P) mutation stabilizing the spike in pre-fusion conformation, a foldon domain and a 10-amino acid linker (10lnQQ) followed by the AVI tag. (B) Structural model of a SARS-CoV-2 spike protomer with the NTD, RBD and SD1 domains highlighted. (C) Sequence of the SARS-CoV-2 spike probe with key regions annotated. See also Table S1.

Figure 3.. Physical Properties, Antigenic Characteristics, and Cryo-EM Structure of a Biotinylated SARS-CoV-2 S2P Probe

(A) Size exclusion chromatography of the biotinylated SARS-CoV-2 S2P probe in PBS buffer. (B) SDS-PAGE of the SARS-CoV-2 S2P probe with and without the reducing agent DTT. Molecular weight marker (MWM) was run alongside the probe. (C) Negative stain EM of the SARS-CoV-2 S2P probe. The 2D-class averages were shown below the wide-field raw EM image in 5-fold magnification. (D) Biotinylation, antigenicity and receptor recognition of the SARS-CoV-2 S2P probe. The level of biotinylation was evaluated by capture of probes at 40 μg/ml onto the streptavidin biosensors. A biotinylated HIV-1 Env stabilized in prefusion closed conformation by DS-SOSIP mutations and containing the same 10lnQQ-AVI tag at the C-terminus was used for comparison. Binding was assessed using 80 μg/ml S2P probe, 20 μg/ml HIV Env and receptors, or antibodies at 500 nM. Error bars represent standard deviation of triplicate measures. Low level non-specific binding of Fab S652–112 to the HIV-1 Env was observed. (E) Cryo-EM structures of biotinylated SARS-CoV-2 S2P probe. Domains are colored as in Figure 2C sequence. The C-terminal residues 1153–1208 plus the foldon, 10lnQQ-AVI tag, and biotin were not visible in the electron density. See also Figure S3 and Table S2.

Figure 4.. Structure-Based Definition of SARS-CoV-2 Molecular Probes Comprising the NTD, RBD and RBD-SD1 Domains

(A) Cryo-EM structure of the NTD domain in the S2P probe determined in this study (Figure 3E), with reconstruction density shown in orange for NTD domain, and gray otherwise. First ordered residue with density (A27) is highlighted with a blue sphere; last residue of NTD domain (S305) is highlighted with a red sphere. (B) Close-up view of the NTD termini. (C) Sequence of NTD domain probe. The sequence is highlighted in orange except for residues 14–26, which are disordered in the cryo-EM structures. (D) Cryo-EM structure of the RBD domain in spike (Figure 3E), with reconstruction density shown in cyan for RBD domain, and gray otherwise. First residue with density (L329) is highlighted with a blue sphere; last ordered residue of RBD domain (G526) is highlighted with a red sphere. (E) Close-up view of the spike RBD termini. (F) Sequence of RBD domain probe highlighted in cyan. (G) Cryo-EM structure of the RBD-SD1 domains in spike (Figure 3E), with reconstruction density shown in green for RBD-SD1 domain, and gray otherwise. First residue with density (R319) is highlighted with a blue sphere; last ordered residue of RBD-SD1 domain (S591) is highlighted with a red sphere. (H) Close-up view of the spike RBD-SD1 termini. (I) Sequence of the RBD-SD1 domain probe highlighted in green. See also Figures S1 and S3 and Tables S1 and S2.

Figure 5.. Physical Properties and Antigenic Characteristics of Molecular Probes Comprising SARS-CoV-2 NTD, RBD, and RBD-SD1 Domains

(A) Size exclusion chromatography of biotinylated NTD, RBD and RBD-SD1 molecular probes. (B) SDS-PAGE of NTD, RBD and RBD-SD1 molecular probes. RBD-SD1 peak 1 appeared to be partially disulfide linked, and was therefore removed from further analysis; RBD-SD1 thus refers to the monomeric peak 2. (C) Receptor binding (left) and antibody binding (right). Both RBD and RBD-SD1 probes bound to the ACE2 receptor and antibody S652–109 while the NTD probe interacted with antibody S652–118 only. Binding was assessed using 5 μg/ml probe and 500 nM receptor or antibody. Error bars represent standard deviation of triplicate measures. Low level non-specific binding of S652–112 Fab to RBD and RBD-SD1 was observed. See also Figure S2.

Figure 6.. Molecular Probes Comprising Specific Mutations for Knocking out ACE2 Interaction with RBD

(A) Structural model of the RBD-ACE2 complex, with inset panels highlighting RBD mutations designed to reduce ACE2 recognition. ACE2 and RBD are shown in cartoon representation and colored blue and cyan, respectively. Arg mutations are shown in stick representation, with potential clashes with ACE2 highlighted with red discs. (B) Schematic and sequence of mutant RBD probes with sites mutated to Arg highlighted in magenta. (C) Size exclusion chromatography of RBDs with mutations altering ACE2 binding. (D) SDS-PAGE of mutant RBD probes with and without reducing agent as indicated. (E) Receptor and antibody binding of mutant RBD probes. All the knockout mutations abolished RBD binding to the ACE2 receptor while retaining binding to both CR3022 and S652–109 antibodies. Binding was assessed using 7.5 μg/ml RBD-L455RA475R, 80μg/ml RBD-L455RG496R, or 15μg/ml RBD-L455RA475RG502R to account for varying biotinylation levels among the mutants. Receptor or antibodies were at 500 nM. Error bars represent standard deviation of triplicate measures. See also Figure S2 and Table S1.

Figure 7.. Validation of Biotinylated Probes

(A) SPR standard kinetic experiments for ACE2 and single-cycle kinetic experiments for Fabs CR3022, S652–109, S652–112 and S652–118 binding over biotinylated spike (top row) and biotinylated-RBD or -NTD (bottom row) loaded onto a streptavidin sensorchip. Black traces represent the experimental data and the red traces represent the fit to a 1:1 interaction model. The error in each measurement represents the error of the fit. (B) Binding of yeast expressing SARS-CoV cross-reactive Fabs to SARS-CoV-2 antigenic probes. HIV envelope-targeting VRC01 Fab was used as a negative control. (C) Sorting of yeast cells encoding antibody transcripts from a convalescent COVID-19 donor with S2P, NTD and RBD-based probes. (D) Sorting of B cells from COVID-19 convalescent PBMCs. (Top row) PBMCs obtained from a SARS-CoV-2-infected subject 75 days after symptom onset were stained with surface antibodies and probes as outlined in the methods to allow for identification and specificity-determination of SARS-CoV-2 spike-specific IgG+ and IgA+ B cells. (Bottom row) PBMCs obtained pre-pandemic from a healthy donor serve as a control for background binding of the probes. See also Figures S4–S7.