Analysing the spike protein of SARS-CoV-2 with 3DM

Bio-Prodict releases suite of 3DM systems to support COVID-19 drug and vaccine R&D, you can read up on that here.

In this post we will demonstrate what are the capabilities of 3DM using an example of the SARS-CoV-2 spike protein. You’ll see how 3DM systems can make protein research faster and more effective, and how they can help you generate hypotheses.

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The spike protein is a large protein located at the surface of coronaviruses and constitutes the infamous crown of the virus. Because the spike protein is exposed it could be easily accessible for human antibodies - and that makes it a good candidate for vaccine research. It is also part of  the coronavirus’s attack mechanism, as it binds to the human ACE2 receptor which then mediates entry of the virus to human cells. 

Let’s have a look at the SARS-Cov-2 spike protein in the context of the whole protein family, can we maybe find some interesting differences between SARS-Cov-2, SARS-Cov, and other family members?

Protein structure 

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The spike protein is a homotrimer, each chain consisting of approximately 1200 residues.  The human ACE2 protein is bound by the top part of the spike. Unfortunately, for now, there are no X-ray structures available for the complex of SARS-CoV-2 spike protein and the human ACE2. However, there are structures of spike proteins from other virus species (e.g. SARS-CoV)  bound to ACE2 - we can use 3DM to transfer information about ACE2 binding from the SARS-CoV proteins onto the SARS-CoV-2 structure and investigate what are the potential differences and/or similarities in ACE2 binding between the two proteins.  

In our spike glycoprotein system we created a hotspot basket of positions that are in contact with the ACE2 receptor in structure 6ACJ. This hotspot basket is accessible to anyone using the system. Using the visualize tool in 3DM we can view these positions on our SARS-CoV-2 structure (6VSB). This is the receptor binding domain (RBD) of the two structures superposed on top of each other:

Blue is the SARS-CoV-2 structure and green is the SARS-CoV structure. The SARS-CoV-2 structure is missing some residues (the chain breaks are indicated with pink dots), but we can infer information about these from the other structure. In general, the binding interface looks very similar in the two proteins, almost all residues that contact the ACE2 protein are conserved (with the exception of position 498 where SARS-CoV has tyrosine and SARS-CoV-2 a glutamine residue). An interesting thing that we see here is that the conserved residue on position 488 forms a cysteine bridge - with a cysteine (position 480) from the loop that’s missing in the SARS-CoV-2 protein. We can have a look at the alignment now, what we see in the closest proteins is that whenever there’s no cysteine on position 488 the whole loop is also missing. So it’s present in both SARS-Cov-2 (though not crystallized in the structure, we only see it from the sequence alignment) and in SARS-CoV, but not for example in Bat SARS-like coronavirus proteins, even the ones that are very close to SARS-CoV-2 in terms of sequence identity.

These cysteines are definitely important for stabilising this loop, can that also be a factor in binding affinity to the ACE2 receptor? That we don’t know, but it’s just one of the hypotheses that you can raise by using 3DM and have it experimentally tested in a lab. Moreover, further into our investigation we do find an article that confirms the loop’s importance for ACE2 binding in SARS-CoV:

The base of this concavity cradles the N-terminal helix of ACE2; a ridge to one side of it, which is reinforced by the Cys467–Cys474 disulfide bridge, contacts the loops between ACE2 helices α2 and α3; a ridge to the other side inserts between a short ACE2 helix (residues 329 to 333) and a β hairpin at ACE2 residue 353 (Fig. 1C). Residues 445 to 460 of the RBD anchor the entire receptor-binding loop to the core of the RBD. We refer to this loop (residues 424 to 494), which makes all the contacts with ACE2, as the receptor-binding motif (RBM).

Fang Li et al. Structure of SARS Coronavirus Spike Receptor-Binding Domain Complexed with Receptor; Science 16 Sep 2005: Vol. 309, Issue 5742, pp. 1864-1868

Our next step of this analysis would be a comparison of sequences with and without the loop (and with and without the cysteine on position 488) and checking if there’s a correlation with evolutionary pressures on different positions.  We’re not going do that right now as it’s just a short demo to showcase the tools in 3DM, but that’s something that you could follow up on after getting access to the 3DM system.

Sequence projection and literature search

Let’s see if we can find out more about spike protein’s mechanism of action from another tool in 3DM - sequence projection. We’re going to visualize family mutations (i.e. mutations that we mined from the literature and were able to map them onto other proteins in the alignment). Position 493 is one of the residues most often described in the literature, which is a good hint that it is an important residue. We can view the abstracts and see if there’s anything of interest for us.

One of the articles, titled “Identification of two critical amino acid residues of the severe acute respiratory syndrome coronavirus spike protein for its variation in zoonotic tropism transition via a double substitution strategy.” mentions in the abstract that:

(...) a two-amino acid substitution (N479K/T487S) of a huSARS-CoV for those of pcSARS-CoV almost abolished its infection of human cells expressing the SARS-CoV receptor ACE2 but no effect upon the infection of mouse ACE2 cells.

Xiu-Xia Qu et al. Identification of Two Critical Amino Acid Residues of the Severe Acute Respiratory Syndrome Coronavirus Spike Protein for Its Variation in Zoonotic Tropism Transition via a Double Substitution Strategy; Journal of Biological Chemistry; 280, 29588-29595

That’s important information - since in the SARS-CoV-2 protein this residue is a glutamine and in SARS-CoV an asparagine (thus, they can make similar interactions), it is likely that such a substitution would have the same effect as in the SARS-CoV protein. Next step from here would be to investigate that second residue described in the article (T487, that’s position 501 in our alignment). It turns out that in the SARS-CoV-2 protein this residue is not  a threonine but an asparagine - that might mean that the virus has found a way around this deleterious substitution, maybe different residues have taken over the role of the 479&487 residues from SARS-CoV?

Maybe you can figure it out with the use of 3DM and further wet-lab experiments! We’ve shown you only a small subset of 3DM’s capabilities - how it can help you generate hypotheses and narrow down possibilities that can later be confirmed in the lab.

If you’re interested in learning more please contact us or have a look at the 3DM walkthrough.