Structural Analysis of SARS-CoV-2 S1 Protein Complex

Protein Expression, Purification, and Crystallization—The gene encoding single chain (VH-linker-VL) antibody 80R (scFv) was cloned into pET22b (Novagen) containing an N-terminal periplasmic secretion signal pelB, and a thrombin-removable C-terminal His₆ tag. 80R was overexpressed in BL21(DE3) cells at 30 °C for 15 h with 1 mm isopropyl 1-thio-β-d-galactopyranoside. Protein was purified by HisBind nickel-nitrilotriacetic acid (Novagen) column and Superdex 200 gel filtration chromatography (Amersham Biosciences) after thrombin digestion.
The gene encoding S1-RBD (residues 318-510) was cloned into vector pAcGP67A (Pharmingen) containing an N-terminal gp67 secretion signal and a thrombin-cleavable C-terminal His₆ tag. It was expressed in Sf9 cells (Invitrogen) with a multiplicity of infection = 5 for 72 h. Similar to 80R, S1-RBD was purified from the media with HisBind nickel-nitrilotriacetic acid and Superdex 200 columns, with thrombin digestion. N-Linked glycosylation was removed by incubation with peptide:N-glycosidase F (New England Biolabs) at 23 °C, as monitored by SDS-PAGE. S1 RBD-80R complexes were formed by mixing the two purified components and isolated by gel filtration with Superdex 200 in 10 mm Tris-HCl, 150 mm NaCl, pH 7.4. Peak fractions were pooled and concentrated to ∼7 mg/ml. For S1-RBD crystal growth, the protein was also concentrated to ∼7 mg/ml.
Crystals grew by the hanging drop vapor diffusion method at 17 °C over ∼21 days. For S1-RBD, 2 μl of S1-RBD was mixed with an equal volume of well solution containing 4% w/v polyethylene glycol 4000, 0.1 m sodium acetate, pH 4.6. For the S1-RBD-80R complex, 2 μl of the complex was mixed with an equal volume of well solution containing 12.5% w/v polyethylene glycol 4000, 0.1 m sodium acetate, 0.2 m ammonium sulfate, pH 4.6.
Data Collection, Structure Determination, and Refinement— X-ray diffraction data were collected at the National Synchrotron Light Source beamline X6A and X29A for S1-RBD crystals, the Stanford Synchrotron Radiation Laboratory beamline 11.1, and at the Advanced Light Source beamlines 5.0.3 and 12.3.1 for crystals of the S1-RBD-80R complex. Glycerol (25%) was used as a cryoprotectant in both cases. All the data were processed with DENZO and SCALEPACK or with the HKL2000 package (8 ). Crystals of S1 RBD adopt space group P4₃2₁2 with unit cell dimensions a = 75.9 and c = 235.8 (Table 1).TABLE 1

Data collection and refinement statistics

	S1-RBD	S1-RBD-80R
Data collection
Cell parameters	a = 75.9, c = 235.9 Å	a = 47.5, b = 175.9, c = 67.6 Å; β = 96.6°
Space group	P43212	P21
Resolution (Å)	2.2	2.3
Total reflections	233011	159047
Unique reflections	36036	51915
Completeness (%)a	99.9 (99.9)	93.8 (87.0)
Average I/σ(I)a	24.7 (2.0)	8.8 (1.9)
Rmergea	0.098 (0.739)	0.145 (0.571)
Redundancy	6.5	3.1
Refinement
Rworkb	0.182 (0.230)	0.248 (0.301)
Rfree (5% data)b	0.213 (0.289)	0.295 (0.391)
r.m.s.d. bond distance (Å)c	0.013	0.009
r.m.s.d. bond angle (°)	1.49	1.22
Average B value	50.0	37.1
Solvent atoms	152	470
Ramachandran plot
Residues in most favored regions	276	631
Residues in additional allowed regions	35	81
Residues in generously allowed regions	3	5
Residues in disallowed regions	0	0

a

Numbers in parentheses correspond to the highest resolution shell (2.28-2.20 Å for S1 RBD; 2.29-2.38 Å for S1 RBD-80R)

b

Numbers in parentheses correspond to the highest resolution shell (2.26-2.20 Å for S1 RBD; 2.29-2.38 Å for S1 RBD-80R)

c

r.m.s.d., root meant square deviation

Crystals of the S1-RBD-80R complex adopt space group P2₁ with unit cell dimensions a = 47.5, b = 175.9, c = 67.6, β = 96.6°. The crystals display a lattice-translocation defect in which a fraction of the layers have a translational offset, resulting in periodic sharp and diffuse rows of reflections (Fig. 1). Similar defects were first described by Bragg and Howells (9 ). Different crystals displayed different degrees of lattice defects, and data merged poorly between crystals. By using a single crystal we were able to collect a data set of good quality with a final R_MERGE = 0.145 and completeness of 93.8% to 2.3 Å resolution. Processing the data required careful optimization of integration profiles and the imposition of a fixed mosaicity (0.45°). Correlation between the offset layers caused the appearance of a strong off-origin peak (65% of the origin) in the native Patterson map at (1/3, 0, 0), indicating that the dislocation occurred along the a* direction. Additional features of the Patterson map were visible at ∼1/10 of the origin peak and provided a measure of the severity of the defect among different crystals. The averaged intensity for the layers of reflections showed a periodic variation that corresponded to the sharp and diffuse layers, and we used the procedure developed by Wang et al. (10 (link)) to correct for the intensity modulation (Fig. 2). We calculated average intensities for individual h layers, and applied a correction to the intensities using Equation 1,
${\displaystyle {\mathrm{I}}_{\mathrm{C}\mathrm{O}\mathrm{R}}={\mathrm{I}}_{\mathrm{M}\mathrm{E}\mathrm{A}\mathrm{S}}/(A+B\mathrm{c}\mathrm{o}\mathrm{s}(2\pi h\mathrm{\Delta }x))}$ where A and B were obtained by least square fitting of the averaged measured intensities. The ratio of the parameters B and A (B/A = 0.65) coincided with the height ratio of the Patterson peak at (1/3, 0, 0), as required by the lattice-translocation theory presented by Wang. The corrected intensity distribution (Fig. 2b) was used for the structure solution and the refinement.FIGURE 1

Diffraction patterns of complex crystal. The complex crystals display a lattice-translocation defect caused by translocations in the crystal packing between neighboring layers along the a* direction. a, a* is nearly vertical, in the plane of the paper, and the defect results in periodic sharp-diffuse-diffuse rows of diffraction intensities (the bottom left quadrant is a zoom-in of the boxed area). b, a* is nearly parallel to the x-ray beam and perpendicular to the paper, and the defect is not evident.

FIGURE 2

h layer intensities before and after correction.a, the lattice defect results in a strong-weak-weak pattern of intensities along h, which were corrected (b) according to the procedure of Wang et al. (10 (link)).

The structure of the S1-RBD-80R complex was determined using the Joint Center for Structural Genomics molecular replacement pipeline (11 (link)), which employs a modified version of MOLREP (12 ), and independently using PHASER (13 (link)), with the S1-RBD domain from the S1-RBD-ACE2 complex and the scFv domain from the scFv-turkey egg-white lysozyme complex (Protein Data Bank code 1DZB) as search models. The asymmetric unit contains two molecules of S1 RBD-80R. The final model includes residues 318-505 (molecule 1) and 319-509 (molecule 2) of S1 RBD and residues 1-245 (molecule 1) and 1-244 (molecule 2) of 80R, and 470 water molecules. No electron density was observed for the artificial poly(Gly/Ser) inter-domain linker. Initial solutions from molecular replacement were subjected to several rounds of refinement with the program REFMAC5 (14 (link)) with simulated annealing in CNS (15 (link)) and manual model rebuilding with programs O (16 (link)) and Coot (17 (link)).
The structure of uncomplexed S1-RBD (which showed no lattice defects) was determined by molecular replacement with PHASER (13 (link)) using S1-RBD from the structure of the S1-RBD-ACE2 complex (Protein Data Bank code 2AJF) as the search model. The asymmetric unit contains two molecules of S1-RBD arranged as a symmetric dimer. The final model includes residues 320-503 of both monomers and 152 water molecules.
Geometric parameters are excellent as assessed with PRO-CHECK (18 ) (Table 1). Final R_WORK/R_FREE values are 18.2/21.3 and 24.8/29.5 for the uncomplexed S1-RBD and the S1-RBD-80R complex, respectively. The higher R values for the S1 RBD-80R complex can likely be explained by the limitations of the lattice defect model and the integration of weak, elongated spots, as discussed previously (10 (link)). Notwithstanding, the final electron density map for the S1 RBD-80R complex is of excellent quality (Fig. 3), and the model-to-map correlation is above 0.9 for most of the residues at 2.3 Å resolution. Coordinates have been deposited in the Protein Data Bank with codes 2GHV (S1-RBD) and 2GHW (S1-RBD-80R complex).FIGURE 3

Stereo 2F_o - F_c electron density map of the S1-RBD-80R complex at the S1-80R interface. S1 and 80R residues are shown in red and blue, respectively, with selected residues labeled. Contour level = 1.5σ.