Higher binding affinity of furin for SARS-CoV-2 spike (S) protein D614G mutant could be associated with higher SARS-CoV-2 infectivity

Abstract

Objective: The coronavirus disease 2019 (COVID-19) pandemic has caused an exponential rise in death rates and hospitalizations. The aim of this study was to characterize the D614G substitution in the severe acute respiratory syndome coronavirus 2 (SARS-CoV-2) spike glycoprotein (S protein), which may affect viral infectivity.

Methods: The effect of D614G substitution on the structure and thermodynamic stability of the S protein was analyzed with use of DynaMut and SCooP. HDOCK and PRODIGY were used to model furin protease binding to the S protein RRAR cleavage site and calculate binding affinities. Molecular dynamics simulations were used to predict the S protein apo structure, the S protein-furin complex structure, and the free binding energy of the complex.

Results: The D614G substitution in the G clade of SARS-CoV-2 strains introduced structural mobility and decreased the thermal stability of the S protein (ΔΔG = -0.086 kcal mol^-1). The substitution resulted in stronger binding affinity (K_d = 1.6 × 10^-8) for furin, which may enhance S protein cleavage. The results were corroborated by molecular dynamics simulations demonstrating higher binding energy of furin and the S protein D614G mutant (-61.9 kcal mol^-1 compared with -56.78 kcal mol^-1 for wild-type S protein).

Conclusions: The D614G substitution in the G clade induced flexibility of the S protein, resulting in increased furin binding, which may enhance S protein cleavage and infiltration of host cells. Therefore, the SARS-CoV-2 D614G substitution may result in a more virulent strain.

Keywords: COVID-19; Furin; G clade; Interatomic binding; Molecular dynamics simulations; S protein; SARS-CoV-2; Thermodynamic stability.

Figure 1

(A) Trimeric structure of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-S) spike glycoprotein (S protein) (Protein Data Bank ID 6VSB). (B) D614G mutant S protein monomer. The red region of the protein depicts the more flexible region of the protein due to the D614G substitution with decreased stability of ΔΔG = −0.086 kcal mol⁻¹ and an increase in vibrational entropy (ΔΔS_Vib) of 0.137 kcal mol ⁻¹ K⁻¹. NTD, NTD, N-terminal domain; RBD, receptor-binding domain.

Figure 2

(A) Suggested hydrogen bonds (dashed red lines) of D614 (S1 domain chain A) with T859 (S2 domain chain B) and D614 and A646 of S1 domain chain A. (B) This hydrogen bond can be disrupted with the D614G substitution, altering the activity of the protein. (C) Modeled furin bound to the S protein RARR site. Furin bound to WT D614 S protein is depicted in green and bound to the D614G mutant is depicted in purple. (D) The dissociation constant K_d for furin bound to D614 S protein (blue) and G614 S protein (red).

Figure 3

Molecular dynamics simulations. Root-mean-square deviation (RMSD) plots for 100-ns simulations of D614 S protein and G614 S protein (A) and D614 S protein–furin and G614 S protein–furin complexes (B). Root-mean-square fluctuation (RMSF) plots of D614 S protein and G614 S protein (C) and D614 S protein–furin and G614 S protein–furin complexes (D).