Structural impact on SARS-CoV-2 spike protein by D614G substitution

Abstract

Substitution for aspartic acid (D) by glycine (G) at position 614 in the spike (S) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) appears to facilitate rapid viral spread. The G614 strain and its recent variants are now the dominant circulating forms. Here, we report cryo-electron microscopy structures of a full-length G614 S trimer, which adopts three distinct prefusion conformations that differ primarily by the position of one receptor-binding domain. A loop disordered in the D614 S trimer wedges between domains within a protomer in the G614 spike. This added interaction appears to prevent premature dissociation of the G614 trimer-effectively increasing the number of functional spikes and enhancing infectivity-and to modulate structural rearrangements for membrane fusion. These findings extend our understanding of viral entry and suggest an improved immunogen for vaccine development.

Fig. 1. Characterization of the purified full-length SARS-CoV-2 S proteins.

(A) The full-length SARS-CoV-2 S protein carrying either D614 or G614 was extracted and purified in detergent n-dodecyl--d-maltopyranoside (DDM) and further resolved by gel-filtration chromatography on a Superose 6 column. The molecular weight standards include thyoglobulin (670 kDa), -globulin (158 kDa), and ovalbumin (44 kDa). Peak I is the prefusion S trimer; peak II is the postfusion S2 trimer, and peak III is the dissociated monomeric S1. The insets show peak fractions that were analyzed by Coomassie-stained SDS-PAGE. Labeled bands are S, S1, and S2. Fr#, fraction number. (B) Binding analysis of fractions of peak I in (A) with soluble ACE2 constructs by BLI. The purified S proteins were immobilized to AR2G biosensors and dipped into the wells containing ACE2 at various concentrations (5.56 to 450 nM for monomeric ACE2; 2.78 to 225 nM for dimeric ACE2). Binding kinetics was evaluated using a 1:1 Langmuir binding model for the monomeric ACE2 and a bivalent model for dimeric ACE2. The sensorgrams are in black and the fits in red. Binding constants are also summarized here and in table S1. All experiments were repeated at least twice with essentially identical results. K_D, dissociation constant (binding affinity); RU, response unit.

Fig. 2. Cryo-EM structures of the full-length SARS-CoV-2 S protein carrying G614.

(A) Three structures of the G614 S trimerrepresenting a closed, three RBDdown conformation; an RBD-intermediate conformation; and a one RBDup conformationwere modeled on the basis of corresponding cryo-EM density maps at 3.1- to 3.5- resolution. Three protomers (a, b, and c) are colored in red, blue, and green, respectively. RBD locations are indicated. (B) Top views of the superposition of the three structures of the G614 S in (A) in ribbon representation, with the structure of the prefusion trimer of the D614 S (Protein Data Bank ID: 6XR8) shown in yellow. The NTD and RBD of each protomer are indicated. Side views of the superposition are shown in fig. S8.

Fig. 3. Cryo-EM structures of the full-length SARS-CoV-2 S protein carrying G614.

(A) (Top) The structure of the closed, three RBDdown conformation of the D614 S trimer is shown in ribbon diagram with one protomer colored as NTD in blue, RBD in cyan, CTD1 in green, CTD2 in light green, S2 in light blue, the 630 loop in red, and the FPPR in magenta. (Bottom) Structures of three segments (residues 617 to 644) containing the 630 loop in red and three segments (residues 823 to 862) containing the FPPR in magenta from all three protomers (a, b, and c) are shown. The position of each RBD is indicated. (B to D) Structures of the G614 trimer in the closed, three RBDdown conformation, the RBD-intermediate conformation, and the one RBDup conformation, respectively, are shown, as in (A). Dashed lines indicate gaps.

Fig. 4. Close-up views of the D614G substitution.

(A) A close-up view of the region near the residue 614 with superposition of the G614 trimer structure in green (CTD2) and magenta (FPPR) and the D614 trimer in yellow, both in the closed prefusion conformation. Residues G614, D614, and two K854s from both structures are shown in stick model. The direction of the three-fold axis of the trimer is indicated. (B) Location of the 630 loop in the S trimer. The 630 loop is highlighted in red, the NTD in blue, the CTD1 in green, the CTD2 in light green, the S2 in light blue, and the FPPR from a neighboring protomer in magenta. The S1-S2 boundary and the nearest ordered residues Thr⁶⁷⁶ from S1 and Ser⁶⁸⁹ from S2 are all indicated. A strand from the N-terminal end of S2, packed in the CTD2, is highlighted in purple. (C) A view showing that the 630 loop wedges between the NTD and the CTD1 and pushes them apart. (D) Packing of the 630 loop against the hydrophobic surface formed by residues Val⁵⁹⁵, Val⁵⁹⁷, Val⁶¹⁰, Tyr⁶¹², Val⁶⁴², and Ile⁶⁵¹ from the CTD2 and Pro²⁹⁵ from the NTD. Residues Trp⁶³³ and Val⁶³⁵ from the 630 loop contribute to this interaction.