D614G Mutation Alters SARS-CoV-2 Spike Conformation and Enhances Protease Cleavage at the S1/S2 Junction

Abstract

The SARS-CoV-2 spike (S) protein is the target of vaccine design efforts to end the COVID-19 pandemic. Despite a low mutation rate, isolates with the D614G substitution in the S protein appeared early during the pandemic, and are now the dominant form worldwide. Here, we explore spike conformational changes and the effects of the D614G mutation on a soluble S ectodomain construct. Cryo-EM structures reveal altered RBD disposition; antigenicity and proteolysis experiments reveal structural changes and enhanced furin cleavage efficiency of the G614 variant. Furthermore, furin cleavage alters the up/down ratio of the Receptor Binding Domains (RBD) in the G614 S ectodomain, demonstrating an allosteric effect on RBD positioning triggered by changes in the SD2 region, that harbors residue 614 and the furin cleavage site. Our results elucidate SARS-CoV-2 spike conformational landscape and allostery, and have implications for vaccine design.

The Impact on Infectivity and Neutralization Efficiency of SARS-CoV-2 Lineage B.1.351 Pseudovirus

“…Moreover, predicted ∆G values between mutant structures of SARS-CoV-2 spike and ACE2 values showed a correlation with the efficacy of infectivity as shown in Figure 1. Given that the D614G has been shown to enhance cleavage efficiency due to substitution on spike conformational diversity [17,18], we next investigated the effect of RBD mutant of the spike on cleavage state of both Wuhan variant and SA variant. To do so, we transduced the seven different mutations of spike pseudoviruses into indicated cell lines namely Caco-2 and Calu-3 (ACE2+ and TMPRSS2+) or Vero (ACE2+), followed by determining the full length:S2 ratio by immunoblotting using an anti-spike antibody.…”

“…These various mutations may provide an avenue for the SARS-CoV-2 to escape from immunity of the current vaccine against COVID19. Because of this, recent SARS-CoV-2 variant B.1.351, particularly those with mutations in the RBM of spike that potentially bring about conformational changes to the spike protein, got attention [1,17,19]. Herein, we show that K417N and E484K spike mutations derived from SARS-CoV-2 B.1.351 variant has a greater affinity for ACE2 as compared to the D614G derived from SARS-CoV-2 Wuhan variant via MD simulation-based predictions (Table 1 and Figure 2).…”

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

“…the RBD-up conformations more frequently than does the D614 trimer (13,29,32), but it is puzzling why the former binds more weakly to recombinant ACE2 than the latter (32). The known S trimer structures indicate that the D614G change breaks a salt bridge between D614 and a lysine residue (K854) in the fusion peptide proximal region (FPPR) (19,33,34), which may help clamp the RBD in the prefusion conformation.…”

The evolutionary making of SARS-CoV-2

“…Of note, the role of the furin cleavage site in conformational masking seems to be different in the SARS-CoV-2 variant carrying the D614G substitution (Isabel, et al 2020; Korber, et al 2020). D614G results in a constitutively more open conformation of spike (Benton, et al 2020; Gobeil, et al 2021; Turonova, et al 2020; Yurkovetskiy, et al 2020). However, upon furin cleavage the D614G variant of spike has been reported to adopt preferentially a conformation where all three RBDs point down, with the consequence that their RBMs are masked and inaccessible for antibodies, but also for ACE2 (Gobeil, et al 2021).…”

“…D614G results in a constitutively more open conformation of spike (Benton, et al 2020; Gobeil, et al 2021; Turonova, et al 2020; Yurkovetskiy, et al 2020). However, upon furin cleavage the D614G variant of spike has been reported to adopt preferentially a conformation where all three RBDs point down, with the consequence that their RBMs are masked and inaccessible for antibodies, but also for ACE2 (Gobeil, et al 2021). It is tempting to speculate about the role of secreted furin (Vidricaire, et al 1993) that may cleave spike even when it is not receptor bound, and of other proprotein convertases able to cleave at the multibasic furin site (Papa, et al 2021).…”