The outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) triggered the coronavirus disease 2019 (COVID-19) pandemic, which had a huge impact on public health, social life and economic activity worldwide.
This virus binds to its target host cell via its spike protein, which engages the host angiotensin-converting enzyme 2 (ACE2). This precipitates a series of conformational and chemical changes that mediate viral entry and infection.
A new study, released as a preprint on the bioRxiv* server, examines the role played by a fatty acid called linoleic acid (LA) that attaches at a binding site near the receptor-binding domain (RBD) of the spike protein. The spike exists as a homotrimer in open or closed conformations. In the former, at least one RBD points upwards.
Earlier studies showed that LA binding reduces spike-ACE2 binding, stabilizing the locked spike conformation. This leads to inhibition of spike-ACE2 receptor binding by blocking the receptor-binding motif (RBM).
Using molecular dynamics simulations to explore the interactions between these molecules, the researchers at the University of Bristol found that the LA binding site shows stable linkages with the spike trimer. It also intensifies the rigidity of the fatty acid binding site, which then creates a ripple effect affecting the N-terminal domain (NTD).
LA binding site closely linked to distant regions
In the current study, the researchers created a model showing the wildtype spike in its un-cleaved locked state, bound to three LA residues at the fatty acid binding site. This is formed by two adjacent spike protomers.
The LA interacts allosterically to several functionally important spike regions, some far away from its binding pocket, disrupting the dynamic interactions between various spike regions and the receptor. These regions include the RBM on the S1 spike subunit near the LA binding site, the furin cleavage site at the S1/S2 interface, and key regions next to the fusion peptide (FP) on the S2 subunit.
Therefore, LA removal triggers a rapid response which propagates rapidly within the spike. The response starts from the fatty acid binding pocket, spreading to the RBD of the unrelated third protomer.
The RBMs being nearest to the LA binding site respond first to its removal, within a tenth of a nanosecond. The RBM is among the most highly variable spike regions and a prime neutralizing antibody target.
It then involves the furin cleavage site of the first protomer and the NTD of the second protomer. The NTD is a superantigen, though it does not bind to the ACE2 receptor. It is linked to the RBD of an adjacent protomer. It also forms a prime antibody target. LA removal revealed that a network of allosteric bonds linked the LA site with the NTD.
Again, the furin cleavage site responded rapidly and strongly to the removal of LA, through a conformation change, along with a fusion peptide-adjacent region distant from the LA binding site. Since the former, found only in SARS-CoV-2 among the SARS-like CoVs, primes the spike protein, its deletion results in an immediate reduction in spike infectivity.
Changes with D614G mutation
The study also shows how the D614G spike mutation causes an altered pattern of conformational response in the RBD and fusion peptide. This single point mutation has been shown to increase infectivity by improving viral entry into the host cell.
D614G, occurring at the protomer interface, allows the spike to display an exposed loop in the middle of the RBM. This may provide a neutralizing antibody target even when the closed RBD prevents virus-receptor interactions.
The D614G mutation may result in increased mobility at the RBM, which in turn may alter the protective glycan coating prolealter the extent of glycan shielding, affecting the degree to which these epitopes are recognized by their corresponding antibodies. The mutation also disturbs various salt bridges and hydrogen bonds that connect the spike monomers in this region.
The result may be the destabilization of the closed RBD conformation, leading to the predominance of an open conformation. The mutation also reduces the symmetry in the magnitude of the response of each of the spike protomers to LA removal.
The greatest effect of the mutation is thus seen in the disruption of signal propagation between the monomers, from the furin cleavage site both upstream and downstream of the fusion peptide, compared to the wildtype virus. These could have functional effects.
What are the implications?
The approach used in this study proved to be useful in identifying allosteric networks between the spike protomers, indicating its relevance in understanding how mutations affect important functional linkages and change the dynamic interactions of the different variants of the SARS-CoV-2 spike.
The results revealed the close inter-relationship of the spike protomers.
For instance, the furin cleavage site is closely linked to some regions distant from the binding site. As a result, a change at the LA-binding area formed by the interface of two protomers is transmitted widely and rapidly through a network of linked structures, far beyond the adjacent regions, to cause cascading changes in the conformation of other protomers.
This network through which these changes are transmitted contains highly mutable regions. Mutations in or near the furin cleavage site or the allosterically linked regions may impact signal transmission to the fusion peptide or neighboring areas. This in turn could affect virus-membrane fusion, as well as altering neutralizing epitopes.
Spike mutations may thus lead to differences in the way the protein responds to LA binding, and to the subsequent chain of allosteric interactions. Molecules that disrupt the network of communications could be a good starting point for the development of drugs against COVID-19.
The researchers write:
The results here show that the D614G mutation alters the allosteric networks connecting the [fatty acid] site to the regions surrounding the FP. There is reduced communication between the monomers in the D614G spike. The response of the D614G spike to LA is also less symmetrical than the wild-type.”
These findings may explain the altered viral fitness found with this variant.
bioRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.
Oliveira, A. S. F. et al. (2021). The fatty acid site is coupled to functional motifs in the SARS-CoV-2 spike protein and modulates spike allosteric behaviour. bioRxiv preprint. doi: https://doi.org/10.1101/2021.06.07.447341. https://www.biorxiv.org/content/10.1101/2021.06.07.447341v1.