D614G mutation alters SARS-CoV-2 spike conformational dynamics and protease cleavage susceptibility 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 analyze the D614G mutation in the context of a soluble S ectodomain construct. Cryo-EM structures, antigenicity and proteolysis experiments suggest altered conformational dynamics resulting in enhanced furin cleavage efficiency of the G614 variant. Furthermore, furin cleavage alters the conformational dynamics of the Receptor Binding Domains (RBD) in the G614 S ectodomain, demonstrating an allosteric effect on the RBD dynamics triggered by changes in the SD2 region, that harbors residue 614 and the furin cleavage site. Our results elucidate SARS-CoV-2 spike conformational dynamics and allostery, and have implications for vaccine design.

Functional importance of the D614G mutation in the SARS-CoV-2 spike protein

“…Zhang et al also made similar observations; full-length S protein was purified and analyzed by size exclusion chromatography (SEC), and the investigators found that while S(D614) eluted in three peaks representing the prefusion S trimer, postfusion S2 trimer, and dissociated S1 subunit, S(G614) eluted in only one major peak comprised 90% of prefusion S trimer [ 49 ]. Similar SEC profiles for S(D614) versus S(G614) were obtained by Gobeil et al, confirming greater S1 retention in S(G614) [ 52 ]. Additionally, Zhang et al provide a mechanistic explanation for reduced shedding in their interpretation of cryo-EM structures of S protein trimers.…”

“…However, sivelestat also appeared to slightly decrease S1 intensity in the context of S(D614), and one must note that observations of spike in cell lysate my not be representative of that of mature virions. An independent group further observed that protease cleavage at the furin site is more efficient in S(G614) compared to S(D614) [ 52 ], which is supported by computational predictions of furin affinity for the two variants [ 53 ]. On the other hand, Daniloski et al observe that S(G614) compared to S(D614) is more resistant to cleavage as determined by amount of uncleaved S in cell lysates and upon digestion with furin in vitro [ 39 ].…”

“…Therefore, the analyzed macromolecular structures were uncleaved and effects of D614G on intermolecular association between S1 and S2 of processed S protein trimers could not be ascertained. Gobeil et al studied the spike ectodomain structure with the native K986 and V987 residues while maintaining the furin-null mutation, and found that D614 spike trimers had similar melting temperature with or without the di-proline mutation [ 52 ]. Cryo-EM structures of S(D614) and S(G614) spike lacking the di-proline mutation did not identify any populations in “two-up” or “three-up” conformation, while they do show an enrichment of the “one-up” conformation in S(G614) [ 52 ].…”

Dynamic Network Modeling of Allosteric Interactions and Communication Pathways in the SARS-CoV-2 Spike Trimer Mutants: Differential Modulation of Conformational Landscapes and Signal Transmission via Cascades of Regulatory Switches

“… 135 , 136 In support of our results, recent cryo-EM structures of SARS-CoV-2 S mutants revealed an allosteric effect that seemingly small D614G changes in the SD2 domain have on RBD dynamics, suggesting that SD2 regions may be involved in modulating RBD transitions between up and down conformations. 137 Indeed, according to our analysis, the communication paths in the open form may often exploit FP and CTD1/CTD2 regions to connect S2 regions with activated RBDs in the up position ( Figure 10 E,F).…”

Cardiovascular protective properties of oxytocin against COVID-19

“…Importantly, application of OXT can alleviate social isolation-induced atherosclerosis and adipose tissue inflammation [ 165 ]. Currently, newly developed specific vaccines face the challenge of continued mutations in SARS-CoV-2 and immune evasion [ 166 , 167 ] while complete social isolation is difficult to achieve, which leave majority of the population remaining under the threats of COVID-19. Thus, strengthening the basic immunologic function is a measure that universally fits our human society, particularly for aged population who have reduced immunologic function and OXT production [ 168 ].…”