Home » The Structural Basis of Ebola Viral Pathogenesis by Dr. Saphire (Transcript)

The Structural Basis of Ebola Viral Pathogenesis by Dr. Saphire (Transcript)

But perhaps more importantly, we can understand something more about proteins themselves. Because evolution has compelled these proteins to be remarkable, to do more with less than other proteins. By studying what these proteins are capable of, we learn something more about the capabilities of proteins and molecular biology in general.

So I’ll show you a few examples. The first one comes from the first step of the virus life cycle. So the first step – the virus has to find and attach to a new host cell. This is achieved by the glycoprotein called GP. Both of these viruses express only one protein on their surface, the glycoprotein called GP and it is solely responsible for attachment and infusion with that cell.

So Ebola virus is filamentous. This is a cartoon of what the virus might look like, it’s got a membrane envelope, that’s green surrounding the nucleocapsid. And studying to the surface of these glycoprotein spikes. Those for Ebola virus form 450 killadalton trimers and they are quite heavily glycosylated.

So the question you might ask is – if this spike is important for attachment and entry, what does it look like and how does it work?

We had to make about 140 versions of this GP to get one that would crystallize well and we had to grow about 50,000 crystals to get one with the [frac bone]. Before we have a structure, we typically think of a protein as a schematic like this with an N terminus and C terminus. This GP is cleaved in the producer cell, with GL2 sub units. A GP1 which mediates receptor binding and GP2 which mediates fusion. So the GP1 has receptor binding domains and the GP2 has heptad repeats that undergo a conformational change and collapse the six-helix bundle driving membrane fusion. Also in GP1 is this unusual mucin-like domain, it’s very heavily glycosylated. Each domain is about 75 killadalton, it’s 3 in the trimer, there is a lot of unstructured protein and carbohydrate.

So this is the crystal structure of the Ebola virus GP. The first one we solved — you can see the 3GP1 subunits in blue and green, these mediate receptor binding and they are tied together at the bottom by the GP2 fusion subunits.

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Now there is something interesting here. When you think about a fusion peptide for Flu or HIV, it’s a hydrophobic peptide that’s tucked up inside the structure. Well here the fusion loop is tacked under the outside like a [flice water]. The one that belongs to this monomer reaches around the outside of the trimer and binds into the next one over. In order to get this molecule of the crystallized, we had to exize that mucin-like domain.

But we want to understand what the real GP looks like on the viral surface. So it’s got these heavily glycosylated domains attached at the top. Well note GP containing that mucin domain crystallizes, so we had to use a different technique which was small angle X-ray scattering, tiny x-rays and protein molecules tumbling around in solution, you get a low resolution view, maybe 10 angstrom resolution. And in this, that it turns out this is the solution scattering envelope of the complete fully glycosylated Ebola virus GP, with all of its sugars and all of its mucin like domains. So the crystal structure I am showing you in the ribbon, in the center for scale, these are the mucin-like domains attached. So they effectively triple the size of the molecule. When Peter Kwong coined the term glycan shield, this is hell of a glycan shield. They reach about 100 angstrom away from the core of GP and they are quite flexible. So I would actually expect the actual width of this domain to be half that, I think we’re visualizing solution, a lot of flexibility in waving our [camera].

The salient feature of this is that these mucin-like domains are massive and they dominate the structure of the GP.

So this is what is on the virus surface. How does it work? How does it find and get into a new host cell? Well, this I’m showing again the crystal structure, I am coloring the surface white, patches that are colored pink, are areas that mutagenesis tells us are important for infectivity. They are a little bit sequestered inside the bowl shape the trimer makes. The rest of these are most important for a receptor binding are very sequestered. In fact, inside structure underneath this domain. So that is sort of a representation of where the mucin-like domains are. The parts that are important for the receptor binding are these pink ones and they are underneath these domains called the glycan cap, these have a lot of glycan attached to them.

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So, does this make sense and how on earth is this a functional receptor binding site underneath this entire canopy of protein and carbohydrates?

The answer is that it is known from biology that GP needs to be cleaved by host capsaicin enzymes for infection to occur. The filovirus is different the requirement for some cleavage but it’s especially important for Ebola virus.

So, why? Well, in solving the crystal structure, we see that all of this structure, the glycan cap and the whole mucin like domain are attached by a single polypeptide tether that connects residue 189 to 213. And that piece of polypeptide is disordered. So something that is disordered in a crystal structure is flexible and it’s moving around. So this looks like a pretty attractive cleavage site.

If proteases were to cleave on that yellow loop, this would be the effect, which is much better exposure of the receptor binding sites. Now we are not making that up. This is actually the crystal structure now cleaved GP and another lap simultaneously shows that yes, cleavage indeed strips off 85% of the mass of GP1 leaving the receptor binding sites exposed.

So this is what the protein looks like on the viral surface. What do we learn from this? Receptor binding probably doesn’t happen at the viral surface. Just by looking at the structure alone, you can see spots needed to bind that receptor are not accessible. They are not well exposed in this kind of protein. Instead, the virus that bears this surface enters cells by macropinocytosis.

Once in the endosome, this is cleaved to strip off all that surface sugar in these mucin-like domains leaving the receptor binding site well exposed and allowing binding by the receptor Niemann-Pick C1, NPC1. And this binding site is right there where the glycan cap used to be.

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