D614G mutation in the SARS-CoV-2 spike protein enhances viral fitness by desensitizing it to temperature-dependent denaturation

Abstract

The D614G mutation in the spike protein of SARS-CoV-2 alters the fitness of the virus, leading to the dominant form observed in the COVID-19 pandemic. However, the molecular basis of the mechanism by which this mutation enhances fitness is not clear. Here we demonstrated by cryo-electron microscopy that the D614G mutation resulted in increased propensity of multiple receptor-binding domains (RBDs) in an upward conformation poised for host receptor binding. Multiple substates within the one RBD-up or two RBD-up conformational space were determined. According to negative staining electron microscopy, differential scanning calorimetry, and differential scanning fluorimetry, the most significant impact of the mutation lies in its ability to eliminate the unusual cold-induced unfolding characteristics and to significantly increase the thermal stability under physiological pH. The D614G spike variant also exhibited exceptional long-term stability when stored at 37 °C for up to 2 months. Our findings shed light on how the D614G mutation enhances the infectivity of SARS-CoV-2 through a stabilizing mutation and suggest an approach for better design of spike protein-based conjugates for vaccine development.

Keywords: D614G mutation; SARS-CoV-2; cold-induced unfolding; cryo-electron microscopy; protein stability; spike protein; thermodynamics; vaccine development; viral protein.

Figure 1

Cryo-EM analysis of S-D614G.A, schematic domain architecture of S-D614G. The regions not resolved in the cryo-EM map are highlighted with dash line. B, cartoon representative of the atomic model of the trimeric S-D614G with the domains colored in accordance with (A). C, structural heterogeneity of S-D614G. Orthogonal views—side views and top views are shown on the top and bottom panels, respectively—of the five distinct clusters of S-D614G derived from 3DVA. The RBDs are colored in blue, and the N-glycans are colored in yellow green. The nominal resolution of the cryo-EM map and the relative population in percentages are shown below each cluster. D, comparison of conformational changes of the RBDs. The structures of one RBD-up and those of two RBD-up were superimposed separately. The RBDs are colored in blue, green, orange, cyan, and magenta for PDB entries 7EAZ, 7EB0, 7EB3, 7EB4, and 7EB5, respectively. The centers of mass (COMs) of individual RBDs are shown in sphere with the matching colors. To calculate the upward rotation angle (θ) of the individual RBD-up conformations, the COM of an RBD-down conformation is generated by aligning the RBD-up protomer structure with respect to the S2 domain of the RBD-up protomer. The hinge is defined as the Cα atom of residue 330 of the RBD-down protomer. The rotation angles (θ) of individual RBD-up conformations are shown in the side views with matching colors. FP, fusion peptide; HR1/HR2, heptad repeat 1/2; NTD, N-terminal domain; RBD, receptor-binding domain; TM, transmembrane domain.

Figure 2

S-D614G is highly stable over a broad range of temperatures. Representative NSEM micrographs of S-D614 (top panels) and S-D614G (bottom panels) after different temperature treatments. Selected 2D classes of picked particle images are shown below each panel. All micrographs and 2D classes are shown in the same scales. The scale bars of the micrograph and the 2D classes are indicated on the lower left corners of the Day 0 datasets. Significant unfolding of S-D614 was observed after heat shocks and after 6 days of incubation at 4 °C. In contrast, S-D614G exhibited visible unfolding only by heat shock at 60 °C for 30 min 3D maps of the individual samples derived from the native-like particle images are shown on the upper right corner of each panel.

Figure 3

Quantitative analyses of the thermal stabilities of S-D614 and S-D614G.A, histograms of the relative amounts of native-like particle images with respect to the fresh samples. The Y-axis corresponds to the number of micrographs (n = 50–60). The open and filled curves correspond to S-D614 and S-D614G, respectively. B, DSC profiles S-D614 (dashed lines) and S-D614G (solid lines) at Day 0 (fresh; black), 37 °C for 6 days (orange) and 4 °C for 6 days (light blue). C, DSF profiles of S-D614 (dashed lines) and S-D614G (solid lines) as a function of pH values. The difference between S-D614G and S-D614 (Diff.) is derived by subtracting the values of S-D614 by those of S-D614G.

Figure 4

Relative native-like particle number of S-D614G as a function of temperature. All samples were incubated at the specified temperatures as indicated along the X-axis for 30 min prior to NESM grid preparation. The numbers of native-like particles in individual micrographs were normalized with respect to the average value of freshly prepared sample (37 °C, day 0). The error bars correspond to the standard deviations of the numbers within the micrographs collected under the same conditions. For each temperature, between 50 and 60 NSEM micrographs were collected. 3D maps of the individual samples derived from the native-like particle images are shown next to the data points.