As promised, here’s part 2 of my talk on the coronavirus Spike (S) protein biochemistry. I go into cleavage sites, glycosylation, and binding (receptor and antibody). See yesterday’s post for part 1. part 1: blog: ; YouTube:

Here’s a bit of text about what I talk about – the text is adapted from a longer post I made you can find here: 

Antibodies are little proteins that specifically bind viral parts, such as viral proteins, like the spike protein. When someone’s infected, their body makes antibodies against the infector by mixing & matching constant & variable regions to find ones that specifically bind parts of the invader. This allows them to call in for backup from other immune system components when they find the virus. And neutralizing antibodies have the added bonus that they bind to the virus in such a way that the virus can’t get into cells at all – thus “neutralizing” the threat. Such neutralizing antibodies are therefore highly valued and you can learn more about them here: ⠀

Neutralizing antibodies often work by binding and thereby blocking the part of a virus that normally binds to the cell in an “I got here first!” fashion. For the spike protein, this means binding the Spike protein’s RBD. Some antibodies bind directly to the RBM (quick reminder since there’s been a lot of acronyms, the RBM is the receptor binding motif, the actual part of the protein that directly binds to the ACE2 receptor). It’s easy to see how these could block entry. But other neutralizing antibodies are sneakier – they bind the S protein elsewhere but keep the protein from shape-shifting. ⠀

One challenge is that the virus doesn’t want your body to find it, so it uses tricks including glycosylation . When your immune system “sees” the spike protein, it doesn’t just see the amino acids, instead, the surface of the protein has a bunch of sugar chains sticking off of it. This sugary shelf helps protect it from our immune system – which, instead of seeing a foreign protein sees the sugars our body uses all the time. This “glycosylation” is a form of “post-translational modification.” Post-translational just means it happens after translation, the process by which protein-making complexes called ribosomes use the virus’ genetic info as a recipe for sticking together the amino acids into chains. After getting chained together, some of S’s amino acids get glycosylated, which means that they have sugar chains latched onto them. ⠀

Each S protomer has 22 potential N-glycosylation sites & 4 O-ones. And, although the glycosylation states found in a few studies vary a little, most, if not all, of potential N-glycosylation sites are indeed glycosylated (though the composition of the different sugar chains can differ)- so you’re looking at around 60 sugars per spike. This glycosylation, in addition to providing that camouflaging glycan shield, also earns the S protein the fancy title of “glycoprotein.” ⠀

Another “title” you may have seen given to the spike protein is “class I fusion protein.” Other viruses use similar strategies for getting into cells – for example, the flu’s hemagglutinin (HA) protein is also in this class. I didn’t know much about these fusion proteins before, but they’re really quite amazing. Here’s a simplified overview. And here’s a link to an article with more details:

A region called the fusion peptide is at their heart – literally! The fusion peptide is hydrophobic – water doesn’t want to hang out with it – so in the pre-fusion state it’s hidden deep inside the protein. But when you cut the top of the protein off and loosen things up, that lipid-loving peptide faces a watery environment, “sees” that nearby cellular lipid membrane and shoots out towards it, latching on, now connected on both ends. This “panic and grab” leaves it in an awkward position, so next up is getting comfy. To do this, it refolds into a newer, more compact & energetically-favorable, shape, pulling the cell membrane with it as it does. When 2 neighboring S proteins do this, they merge. Kinda like this = -> )( -> – . This merging dumps out the viral contents into the cell, where the virus can go to work getting our cells to go to work for it – making new copies of the virus and shipping them out.

Since the Spike protein is the virus’ key to cellular entry, it’s been getting a lot of interest from a genomic and evolutionary level. You know how I said that S gets cleaved to trigger the major shape-shift where the fusion peptides shoot out? With the original SARS virus, this cleavage occurs by a plasma membrane-bound protease called TMPRSS2, which can do the cleavage early on, while the virus is still at the cell surface, or by a protease called cathepsin L, which can cleave it later, when the virus is “swallowed” into a membrane-bound pouch called an endosome – such “endocytosis” is kinda like the cell pinches in the part of the plasma membrane containing the receptor-bound spike protein, giving you a little membrane-bound “quarantine” bubble inside the cell that the virus now has to escape from. The escape is aided by a protease called cathepsin which is activated by the low pH (acidic-ness) of the endosome & can cleave the Spike protein to enable fusion. Both of those proteases cleave a site called S2’, which all coronaviruses have, and which is crucial to allowing for fusion. ⠀

That’s one of 2 cleavage sites – and the other one’s getting more attention. This “other site” is called S1/S2 because it’s at the border of the S1/S2 domains. And one of the first findings that scientists made about SARS-CoV-2 after sequencing its genome (genetic blueprint) is that it has an “extra” letters at this second site, and those letters are ones a protease called furin likes. ⠀

 Next to the S2’ site is a “polybasic cleavage site” referred to as S1/S2. Basically, polybasic (aka multibasic) means that there are a lot of usually-positively-charged protein letters (amino acids) next to each other – in the case of SARS-Cov2, there’s the sequence RRAR. And this type of sequence can serve as a cleavage site for a protease called furin. They thought this could be important because the furin protease is much more “ubiquitous,” meaning that it’s expressed in a wider variety of cells, therefore making it easier for SARS-CoV-2 to infect various cells. ⠀

Scientists have now done a series of experiments showing that this furin site is important, but it’s not a major advantage in many cases. Basically, the furin cleavage at S1/S2 occurs while a new spike protein is being made. And it kinda loosens things up to make it easier for S2’ to be cleaved, which can make a difference in cells that don’t have a lot of TMPRSS2, but it doesn’t make a big difference if the cells do have a bunch of TMPRSS2.⠀

Some people have claimed that this polybasic cleavage site is evidence of viral man-made-ness. But scientists were quick to point out that 1) not only had this exact sequence ever been seen before, 2) it isn’t “ideal” – so “makers” 1) wouldn’t have just been able to “plagiarize” another virus and 2) wouldn’t have just made up this sequence when they could have made up something better.⠀

Speaking of better, I hope you now have a better understanding of Spike!⠀

Here’s the info for the papers whose structures I showed you:

Cryo-EM structure of the 2019-nCoV spike in the prefusion conformation


SCIENCE13 MAR 2020 : 1260-1263  


SCIENCE25 SEP 2020 : 1586-1592

more Covid-19 resources:

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