Image: Figure 4A, R. Yan et al., Science 10.1126/science.abb2762 (2020).
The paper that we’re demystifying can be found here, if you want to follow along.
These unsung heroes who published this paper in Science on March 9 2020 are a group of scientists from the Key Laboratory of Structural Biology of Zhejiang Province in the Institute of Biology and School of Life Sciences both from Westlake University, Hangzhou China; and Beijing Advanced Innovation Center for Structural Biology at the Tsinghua University. These guys were one of the 1st groups to visualize ACE2 with SARS-CoV-2 spike glycoprotein and I think they helped paved the way for a better understanding on how we can develop mechanisms to defeat this virus.
Before I dive into this paper, I want to explain something that is essentially Virology 101. Viruses are made up of 3 essential components:
1. Their nucleic acid genetic code, it can either be DNA or RNA
2. Their protein capsid
3. Their spike glycoproteins (sometimes known as virus attachment proteins)
A really nice way to visualise this: is a raw chicken egg. The egg yolk is the genetic material, the shell is the protein capsid, and, bare with me here, imagine tiny little spike proteins sticking out of the eggshell, those are the spike glycoproteins.
Now some viruses are unique and may have a fourth additional component. This is known as the envelope membrane. The envelope membrane surrounds the capsid and is usually stolen from the host cell phospholipid bilayer. This bilayer is essentially a type of fat that has amphipathic characteristics (meaning one side is water lover, and the other side is water hating). Clever clever, the phospholipid bilayer basically has 2 rows of these amphipathic fats, so that the inside with which water hating will never touch water, but the outsides are water loving, so they are always in contact with water (I will talk a little more about this later).
In our egg analogy: think of the envelope membrane as grape jelly that has been smeared all over the shell. Gross I know, but it helps paint the picture. Our imaginary spike proteins are still sticking out of the shell and through the grape jelly as well.
Now obviously, the grape jelly is in no way made by the egg, but it’s there; and for our typical enveloped viruses, the envelope membrane is not made by the virus, but was stolen from the cell it invaded.
So that’s Virology 101. But to explain this paper, you also need a quick Cell Biology 101 crash course. Complicated, I know. But allow me to explain, all animals, insects, and even yeast cells are made up of a few important components that are essential for life.
A cell contains many different components that can be considered the organs of the cell, in biology, we call these components organelles. I’m sure many of you heard the “mitochondria is the powerhouse of the cell”. The mitochondria we all love so much, is an organelle.
But I want to touch focus on 1 component of the cell, the cell membrane (otherwise known as the plasma membrane). This bad boy is a phospholipid bilayer. Like I said earlier, a phospholipid bilayer has 2 sides that are water loving. The plasma membrane has one very important job, to keep the things outside of the cell: outside, and the stuff inside of the cell: inside. This is done with the aid of phospholipid bilayer. One side of the water loving layer interacts with the outside of the cell, while the other side interacts with the inside of the cell.
Like your room, the walls are like the plasma membrane. It keeps the outside (your kitchen pot for example) outside of your room, but keeps you and the things in your room, inside your room. But you can freely get up and leave your room through your door.
Well let me tell you something else, the plasma membrane also has things like a door and controls what can come in or leave the cell. The plasma membrane can control what goes in, what goes out, and even relay messages to the rest of your cells by these things known as integral proteins. These proteins are attached to the outside or the inside of the plasma membrane and each one has their own independent roles.
Now what I want you to know is key, some of these proteins that are bound to the outside of the plasma membrane can bind to external proteins which then leads to those external proteins walking through the door and into the cell. These proteins bound to the outside of the plasma membrane are known as receptor proteins.
So this is where things get pretty wild. I told you about how viruses have spike glycoproteins and now you know that cells have receptor proteins. Well, these things go hand in hand. A virus’ spike glycoproteins have the ability to bind to unique protein receptors found only on certain cells. This is why an influenza virus infects cells found in the respiratory tract. The influenza spike glycoproteins only recognise a specific type of receptor proteins found in cells that belong in the upper respiratory tract.
That’s the crash course, now let’s talk about the paper.
The paper starts off by describing the basics of the SARS-CoV-2 virus. This virus is a positive-strand RNA virus and that it can cause severe respiratory syndrome in humans. It goes on to explain that the spike glycoprotein (often depicted as “S protein”- I’ll be honest, virologists are not that creative when it comes to names) is key to recognising the correct protein receptor.
Now, the paper goes on to give a brief history check on SARS-CoV, the virus that caused the 2003 SARS outbreak. It goes on to explain that the S protein of SARS-CoV is a homotrimer protein, meaning that three (tri) identical (homo) proteins are put together. The SARS-CoV S protein homotrimer can be cut into 2 parts: the S1 protein subunit and the S2 protein subunit. The S1 protein subunit contains a key structure known as the Receptor Binding Domain (RBD) that recognises and binds the host (cell) protein receptor, which was identified to be ACE2 (known as Angiotensin-converting enzyme 2). The S1 receptor binding domain recognises a specific part of ACE2, known as the peptidase domain. The S2 subunit is responsible for fusing the SARS virus envelope membrane to the cell plasma membrane.
When the S1 receptor binding domain binds to the peptidase domain of ACE2, the virus is very sneaky and uses an enzyme found in the human body to expose yet another cut site on the S2 protein subunit. This is crucial for viral infection to take place in a cell.
The authors of this paper wanted to visualise how SARS-CoV-2, the virus that is causing the COVID-19 pandemic, S protein which also has the S1 and S2 protein subunit can bind to the peptidase domain of ACE2.
They go on to explain how ACE2, the protein receptor which can be found on the plasma membrane of our lung cells, heart cells, and small intestine cells, job is to control blood pressure and explain the type of integral protein it is. This isn’t too important for understanding their work, but they wanted to showcase how complicated the ACE2 receptor is and its association with cardiovascular diseases among other things.
They do explain another function of ACE2 with the small intestine cells is to be a chaperone for a type of amino acid transporter (the building blocks of all proteins). This amino acid transporter is known as B0AT1. It’s not important to understand what this does, but it is important to understand that these scientists were able to visualise the full structure of ACE2 with the presence of B0AT1. This is quite cool, because we never were able to properly determine the whole structure of ACE2.
Now how did they even visualise such a tiny protein that we can’t even see?
Well let me tell you an inside secret, we use this brand new technology called: cryogenic electron microscopy (cryo-em). It’s a complicated mechanism and you don’t have to understand how it works, but understand that this hunk allows for us to visualise proteins with great resolution. So let’s dive into the results.
- Determining the structure of ACE2
So the scientists first had express ACE2 using a standard set of cells that we can grow in the lab. After these cells were able to express the receptor proteins, they then removed the cells and only kept the ACE2 protein through a series of purification steps. The now purified ACE2 protein was prepared using a standard recipe that the scientists designed to visualise the structure of the whole protein using cryo-em. By visualising the whole ACE2 protein structure the scientist noticed that ACE2 is a heterodimer, meaning two (dimer) differently shaped (hetero) protein stuctures put together.
By capturing images of the ACE2 protein structure in a high resolution, the scientists were able to identify different components that make up the ACE2 protein along with the peptidase domain that has previously been identified.
One of the components that the noticed was of importance was the “Neck” domain which ensures that ACE2 becomes a heterodimer with the peptidase domain contributing a small surface that contributes to forming the ACE2 structure. They identified that these sorta kinda charged building blocks of proteins interact in a specific way that allows for the 2 components of ACE2 to form its proper structure. The peptidase domain, which if you remember is involved with binding to the SARS-CoV and SARS-CoV-2 spike glycoprotein S1 subunit, forms a much weaker sorta kinda slightly charged interactions to ensure that it remains in an open conformation. This essentially means that the peptidase domain keeps a particular shape to make sure that external proteins can bind to it to allow for downstream message relays to the cell.
- Looking at the receptor binding domain of S1 protein to ACE2
So to look at how SARS-CoV-2 S protein receptor binding domain, which if you remember is found on the S1 part of the S protein, the scientists followed another recipe that they designed and once again visualised the receptor binding domain interaction with ACE2. they saw that ACE2 turned into its closed form.
What you should know is that receptor proteins usually have two structures, an open conformation and a close conformation. This essentially means that in an open form, the receptor protein is ready to bind to an external protein, and in a closed form, it is bound to an external protein and cannot bind to anything else at the moment.
So when the S1 receptor binding domain of the SARS-CoV-2 S protein binds to ACE2, ACE2 turns to its closed form. The paper goes on to explain some pretty complex biochemistry that goes on when the receptor binding domain interacts with ACE2’ peptidase domain. I won’t go into details since it’s not necessary for understanding this paper.
- Comparing SARS-CoV-2 and SARS-CoV interaction with ACE2
So when cryo-em is performed we essentially end up taking a lot of pictures of the protein structure we decided to look into. These photos can be superimposed on top of each other and we can look at similarity of protein structures.
The scientists here essentially did this by superimposing the SARS-CoV-2 receptor binding domain with the SARS-CoV receptor binding domain and found the two to be super similar to each other. But despite the high similarity they also noticed that there is a fair amount of differences between the 2 conformations indicating slight differences in the amino acid code that makes up the receptor binding domain. This essentially does affect how the 2 different receptor binding domains interact with ACE2.
So what does all of that result in for future work?
Well these scientists were able to show how ACE2 is a dimer and may actually be a homodimer (meaning 2 of the same protein structure put together) rather than a heterodimer. It also showed that SARS-CoV-2 receptor binding domain that interacts with ACE2 has some changes (as a result of mutation- see post here for explanation on mutations). These changes could potentially make the SARS-CoV-2 S1 interaction with ACE2 stronger or even weaker depending on what specific change they looked at.
All in all what this team of scientists were able to show could aid in other scientists’ ability to understand and develop new mechanisms for treating and suppressing SARS-CoV-2 from infecting cells that contain the ACE2 protein receptor.