In a series of important papers, Gamblin’s postdocs Antoni Wrobel and Donald Benton co-led projects in which they described the structure of the SARS-CoV-2 spike (S) protein and compared it to that of its closest relative, a bat coronavirus (Wrobel et al, 2020), and then generated high-resolution cryo-EM structures showing the process by which SARS-CoV-2 S protein unfurls and docks with its receptor, human ACE2 (Benton et al, 2020). Most recently, they have demonstrated that at least part of the enhanced infectivity of SARS-CoV-2 variants carrying the D614G mutation is likely due to a more open spike structure which is primed for binding to ACE2, and perhaps also for membrane fusion (Benton et al, 2021). This in-depth understanding of spike structure not only provides a direct insight into the mechanism of infection but also provides invaluable information for the design of anti-spike therapeutics.
Infection by SARS-CoV-2 is initiated by virus binding to ACE2 cell surface receptors, followed by fusion of the virus and cell membranes to release the virus genome into the cell. As described above, both receptor binding and viral fusion are mediated by the viral spike (S) glycoprotein, which is post-translationally cleaved by furin into S1 and S2 components that remain associated after cleavage. When the SARS-CoV-2 spike protein is compared to that of the bat virus RaTG13, its closest known relative, this furin cleavage site is missing. This is of significance, as similar furin cleavage sites have been found in related coronaviruses, including MERS-CoV, and their acquisition is associated with increased pathogenicity in other viruses such as influenza virus.
Previous structural studies of the spike glycoproteins of other coronaviruses have shown that the spike is a trimer, consisting of a central helical stalk — comprising three interacting S2 components — capped by the S1s. Each S1 component has two large domains, the N-terminal domain and the receptor-binding domain (RBD), with each associated with a smaller intermediate subdomain. Following purification and furin cleavage, Wrobel et al (2020) derived cryo-EM structures for the SARS-CoV-2 spike protein trimer, and found that they could distinguish three conformations: closed, intermediate and open. In the closed conformation, the RBDs were buried, with the ACE2 binding interfaces inaccessible inside the trimer, but in the intermediate form, one of the RBDs was now disordered; this disordered RBD was found rotated by ~60° in the open form so that the ACE2-interacting surface was fully exposed at the top of the assembly.
In furin-cleaved material, a much lower proportion of spike trimers were in the closed conformation, suggesting that as well as its requirement for membrane fusion, efficient cleavage might be selected to ensure there is a higher proportion of spike protein on the virus surface in an open conformation capable of receptor binding. Further evidence for this hypothesis came from a comparison of the cryo-EM structures of the SARS-CoV-2 and RaTG13 spike proteins. Mutations in the RBD-RBD interfaces of the S1 part of the trimer mean that the closed, uncleaved SARS-CoV-2 spike trimer is more stable than that of RaTG13, but once cleaved, the SARS-CoV-2 trimer has similar stability to its bat counterpart.
By using surface biolayer interferometry, the group also measured receptor binding of the SARS-CoV-2 and RaTG13 spikes. Their data showed that when compared to the RaTG13 spike protein, the changes present in SARS-CoV-2 allowed 1000- fold tighter binding to the ACE2 receptor, meaning that the RaTG13 spike would be unable to bind effectively. The RaTG13 virus is therefore unlikely to have ever infected humans directly; rather, the observations suggest the involvement of recombination between distinct coronavirus genomes in the generation of SARS-CoV-2.
Benton et al (2020) built on this work to generate a detailed picture of the binding of the SARS-CoV-2 spike trimer to the ACE2 receptor. Using cryo-EM, they were able to classify ten different molecular species on a spectrum from the unbound, closed spike trimer through the fully open ACE-2 bound trimer, to dissociated monomeric S1 bound to ACE2. Taken together, these structural data enable mechanistic suggestions for the early stages of SARS-CoV-2 infection of cells.
As shown in Wrobel et al (2020), after furin cleavage between the S1 and S2 domains, the proportion of spike trimers in an open conformation with one erect RBD capable of binding ACE2 increases. Binding of the ACE2 receptor to an open RBD is incompatible with the RBD adopting a closed conformation, leading to several two-open-RBD conformations as well as the three-RBD-bound conformation. Successive RBD opening and ACE2 binding generates the fully open and ACE2-bound form in which the trimeric S1 ring remains bound to the core S2 trimer by limited contacts through the intermediate subdomains of S1. Once in this arrangement, the tops of the S2 helices are fully exposed, and the S trimer is primed for the helical rearrangements of S2 that are required for fusion of the viral and host cell membranes. By analogy with other coronaviruses, the fusion peptide is then projected towards the host cell membrane, a structural change that is driven by the cooperative displacement and dissociation of the three S1 monomers.
This work used the original version of the SARS-CoV-2 spike protein, but during 2020, a mutation, D614G, disseminated rapidly through the population to become the most abundant variant worldwide; this D614G mutation remains in all emerging variants including the highly infectious Kent and South African strains. To determine if there was a structural reason for the variant’s increased infectivity, Benton et al (2021) compared the cryo-EM structures of the G614-substituted spike protein with the D614 original. They found that unlike its progenitor, which is predominantly closed, the G614 spike protein adopts a predominantly open conformation. Therefore, G614 promotes opening of the spike, priming it for binding to ACE2 and possibly for its subsequent role in membrane fusion. This open conformation may be the reason for the current virus’s reported increased infectivity and its current predominance.
While a worrying development, the G614 spike mutant may have an Achilles heel: during the first wave of infection, most people did not have antibodies to SARS-CoV-2, so the open-structured G614 spike protein could have been beneficial to the virus. However, as time goes on, more people will have antibodies as a result of previous infection or vaccination. As the mutant spike protein presents more surface area, it is more exposed to these antibodies, which could potentially be a disadvantage.