Network analysis uncovers the communication structure of SARS-CoV-2 spike protein identifying sites for immunogen design

Abstract

The COVID-19 pandemic, caused by the SARS-CoV-2 virus, has triggered myriad efforts to understand the structure and dynamics of this complex pathogen. The spike glycoprotein of SARS-CoV-2 is a significant target for immunogens as it is the means by which the virus enters human cells, while simultaneously sporting mutations responsible for immune escape. These functional and escape processes are regulated by complex molecular-level interactions. Our study presents quantitative insights on domain and residue contributions to allosteric communication, immune evasion, and local- and global-level control of functions through the derivation of a weighted graph representation from all-atom MD simulations. Focusing on the ancestral form and the D614G-variant, we provide evidence of the utility of our approach by guiding the selection of a mutation that alters the spike's stability. Taken together, the network approach serves as a valuable tool to evaluate communication "hot-spots" in proteins to guide design of stable immunogens.

Keywords: Biological sciences; Immunology; Structural biology; Virology.

Graphical abstract

Figure 1

Structure and graph representations of the SARS-CoV-2 spike protein Top and bottom left panels illustrate the graph and structure representations, respectively, of the spike protein divided into pre-assigned protomer domains (colored regions/nodes). In the bottom right panel, we provide the full domain names, the domain abbreviations, and the residues that comprise them. Each amino acid residue of the protein forms a node in the graph representation. All molecular images were created using VMD (https://doi.org/10.1016/0263-7855(96)00018-5). The graph was drawn using the interface IGraph/M for Wolfram mathematica (https://doi.org/10.5281/zenodo.1134932).

Figure 2

Communication center of the spike protein (A) SARS-CoV-2 spike protein in the structure representation highlighting the NTD supersite (green), alongside the top-50 residues with highest betweenness centrality for the D form only (orange), the G form only (blue), and both forms (black), for the all-down (left panel) and one-up (right panel) conformations. (B) Betweenness centrality broken down by domain for the D and G form and the one-up and all-down conformations. (C) Ranked betweenness centrality for individual residues of the D form (left) and the G form (right) and the all-down (red) and one-up (green) conformations.

Figure 3

Closeness centrality highlights the NTD, CT0, CT1, and CT2 domains (A–C) Structural representation of the SARS-CoV-2 spike protein in the one-up conformation of the G forms colored by the closeness centrality. This measure is also presented broken down by protein region (B) and by individual residues ranked from highest to lowest (C), for the D and the G form, and for the all-down and the one-up conformations.

Figure 4

Eigenvector centrality highlights dominant role of NTD (A) Graph representation of the SARS-CoV-2 spike protein in the one-up conformation of the G form colored by individual node eigenvector centrality. (B) Structure close-up for the NTD and neighboring RBD region with eigenvector centrality highlighted by residue. (C) Domain-level eigenvector centrality for the D and the G forms, and for the all-down and the one-up conformations.

Figure 5

Optimal pathways to RBM from residue 614 and furin cleavage sites SARS-CoV-2 spike protein in the structure representation highlighting the optimal intra-chain (left panel) and inter-chain (right panel) pathways from the furin cleavage (residue 685) to the RBM region (residue 501) for the all-down only (red), one-up only (green), and both conformations (yellow) in the G form. The protomers are labeled R, L, and U. Protomers R and L are set in the down state, while the up or down state of the U protomer defines the conformation of the spike as one-up or all-down, respectively.

Figure 6

Relevance of sites modified/deleted by the Delta variant (A) Structural representation of the SARS-CoV-2 spike protein in the one-up conformation of the G-form highlighting the residues altered in the Delta variant. Deletions are marked in bold font and the $\Delta$ symbol. (B) Analogous to (A) but in the graph representation of the spike protein. (C) Left panel shows the closeness centrality of individual residues ranked from highest to lowest in the one-up conformation of the spike protein. The horizontal grid lines mark the values of closeness associated to the residues modified/deleted by the Delta variant. The colors match the table in the right panel, and shows detailed information concerning the individual ranking position, the identity of the residue (bold font indicates deletions), and the value of closeness in units of ${10}^{-4}$. (D) Average closeness centrality and fluctuations (error bars) of the NTD mutation sites of the Delta variant compared to that of the NTD supersite for the all-down and the one-up conformation.

Figure 7

The N317P mutation alters the SARS-CoV-2 spike RBD disposition (A) Binding of ACE2 to the 2P and N317P 2P spike ectodomains of the ancestral form of the virus. Error bars are standard deviation. (B) Differential scanning fluorimetry profiles for triplicate measures of the 2P and N317P 2P spikes. (C) Negative stain electron microscopy 3D reconstructions of the 1-up and 3-down states of the 2P spike indicating the relative proportion of particles assigned to each state. (D) Negative stain electron microscopy 3D reconstructions of the 1-up, 1/2-up, and 3-down states of the 2P-N317P spike indicating the relative proportion of particles assigned to each state. (E) Refined negative stain electron microscopy 3D reconstruction of the 2P 1-up state with fit coordinates (PDB ID). (F) Refined negative stain electron microscopy 3D reconstruction of the 2P-N317P 1/2-up state with fit coordinates (PDB ID). Red circles indicate position of coordinates outside density.