D936Y and Other Mutations in the Fusion Core of the SARS-CoV-2 Spike Protein Heptad Repeat 1: Frequency, Geographical Distribution, and Structural Effect

Abstract

The crown of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is constituted by its spike (S) glycoprotein. S protein mediates the SARS-CoV-2 entry into the host cells. The "fusion core" of the heptad repeat 1 (HR1) on S plays a crucial role in the virus infectivity, as it is part of a key membrane fusion architecture. While SARS-CoV-2 was becoming a global threat, scientists have been accumulating data on the virus at an impressive pace, both in terms of genomic sequences and of three-dimensional structures. On 15 February 2021, from the SARS-CoV-2 genomic sequences in the GISAID resource, we collected 415,673 complete S protein sequences and identified all the mutations occurring in the HR1 fusion core. This is a 21-residue segment, which, in the post-fusion conformation of the protein, gives many strong interactions with the heptad repeat 2, bringing viral and cellular membranes in proximity for fusion. We investigated the frequency and structural effect of novel mutations accumulated over time in such a crucial region for the virus infectivity. Three mutations were quite frequent, occurring in over 0.1% of the total sequences. These were S929T, D936Y, and S949F, all in the N-terminal half of the HR1 fusion core segment and particularly spread in Europe and USA. The most frequent of them, D936Y, was present in 17% of sequences from Finland and 12% of sequences from Sweden. In the post-fusion conformation of the unmutated S protein, D936 is involved in an inter-monomer salt bridge with R1185. We investigated the effect of the D936Y mutation on the pre-fusion and post-fusion state of the protein by using molecular dynamics, showing how it especially affects the latter one.

Keywords: COVID-19; infectivity; molecular dynamics; mutations; spike protein.

Figure 1

Structural and sequence location of the reported mutations. Top: Cartoon representation of SARS-CoV-2 S protein HR1 and its fusion core (insets) in the pre-fusion and post-fusion conformations (PDB IDs: 6VSB and 6LXT). Discussed mutations are colored in purple and labelled. Q949, at the end of the fusion core, is also labeled. Bottom: Sequence alignment of the HR1 fusion core (framed) and 10 residues up-stream and down-stream in the S protein of SARS-CoV-2, bat coronavirus RaTG13 (protein_ID: QHR63300.2), and SARS-CoV (protein_ID: AAP13441.1).

Figure 2

Countries and genetic clades. Pie chart visualization of the geographical distribution (left panel) and phylogenetic classification (right panel) of sequences presenting the S929T, D936Y, and S939F mutations.

Figure 3

Mutants in the pre-fusion conformation. Right: Cartoon representation of the SARS-CoV-2 S protein in its pre-fusion trimeric conformation (the three monomers are colored in silver, gold, and copper, PDB ID: 6VSB), with the structure of the RBD bound to the ACE2 receptor (in blue, PDB ID: 6M0J) superimposed on its chain A. The most frequent mutations in the HR1 fusion core in GISAID on 15 February are colored purple and shown as a “dots” representation for chain A. Left: Focus on the structural context of each wild-type residue (silver sticks) and corresponding mutant (purple sticks).

Figure 4

Mutants in the post-fusion conformation. Right: Cartoon representation of the SARS-CoV-2 S protein in its post-fusion trimeric conformation (the three monomers are colored in silver, gold, and copper, PDB ID: 6LXT). The color code is the same in Figure 3. Mutations in the HR1 fusion core are shown in a “dots” representation for chain A. Left: Focus on the structural context of each wild-type residue (silver sticks) and corresponding mutant (purple sticks). H-bonds are shown as red, dashed lines.

Figure 5

Comparative MD analysis of the Wuhan reference S protein and the D936Y mutant. (a) rmsd difference (rmsd) between the wild-type and the mutant in the pre-fusion (black) and post-fusion (red) conformation, averaged over the three independent 500-ns simulations per system. (b) Wild-type: minimum distance over time between the carboxylate oxygens of D936 and the guanidinium nitrogens of R1185 on the adjacent monomer, averaged over the three independent simulations per system. Values per single trimer interfaces are plotted as dashed lines while the average values over the three interfaces are plotted as a continuous red line. (c) Mutant: minimum distance over time between the hydroxyl oxygen of Y936 and the guanidinium nitrogen of R1185 on the adjacent monomer, averaged over the three independent simulations per system. Values per single trimer interfaces are plotted as dashed lines while the average values over the three interfaces are plotted as a continuous red line. (d) Mutant: distances over time between the center of mass of the Y936 aromatic ring and the guanidinium nitrogen of R1185 on the adjacent monomer, averaged over the three independent simulations and the three interfaces.