Background Information
The SARS-CoV-2 spike protein is a key protein in the virus life cycle. The spike protein interacts with host cell receptors. In the case of the SARS-CoV-2 coronavirus, that receptor is ACE2, a membrane-bound protein commonly found on the surface of many types of epithelial cells all around the body, particularly in the respiratory tract. The viral spike protein binds to the host ACE2 protein when the virus encounters a receptive host cell. This binding triggers a complex series of protein interactions and rearrangements of spike protein tertiary and quaternary structure which results in the fusion of the viral lipid-bilayer envelope to the cell membrane and entry of the viral RNA genome into the host cell. The virus at this point now has access to the cellular machinery and will begin the biochemistry requireed for viral protein synthesis and genome replication.
Structure
Take a look at the complex structure on the right (you can press the spin button to stop roation of the molecule. You can manipulate the orientation of the molecule by click-dragging in any dimension. You can control the zoom level by using the middle wheel of your mouse (if you have one), spreading apart or bring two fingers together on your touch pad or touch screen.
1. How many polypeptides are shown in this structure? (Hint: count the number of differently colored units)
2. The spike protein is a homotrimer, made up of three identical subunits as you learned from the introduction to this lab. What are the colors of the three subunits that make up the homotrimer?
3. What color is the ACE2 receptor protein?
If you look carefully, you'll notice that the receptor-binding domain (RBD) of one of the three subunits (green) of the spike protein has projected out to interact with ACE2 molecule. This is a conformational change that the spike protein actually makes as it makes contact with the ACE2 receptor, and requires the action of a host-cell protease (an enzyme that cuts peptide bonds in proteins), known as furin, which cuts one of the spike protein subunits. Once cut, the RBD can rearrange into the projected-form, which allows a tight interaction between the spike protein and the ACE2 receptor. This conformational change can be seen in animated form to the right:
Let's focus in on the projected RBD domain. Click on the green link for a close up view of that RBD in isolation.
4. In this representation, what color is used for the alpha helices?
5. How many alpha helices are there in this structure?
6. What color is used for beta sheets?
Now, let's add the ACE2 receptor to the scene. Click on the link to see the RBD interaction with the ACE2 protein.
3. Which subunit(s) (in the 3D model) is/are the MHC molecule? Which subunit(s) is/are the peptide? Think of the hot dog and bun analogy...
4. Which subunit(s) of the MHC molecule play a direct role in binding of the peptide?
- 5. The fit of the peptide in the MHC molecule requires some precision and the peptide must be within a narrow range or lengths. Why do you think this might be (think about the consequences if just any old peptide (of any length) could fit into the MHC groove)?
Hmm...but just how tight does the fit of the peptide look in the MHC groove? Now, open the . Here you are seeing only subunits D and F from the previous image.
6. What is different - or - where did all the space go? To get a feel for this new view and how it relates to the previous model, click on the "popup" button on the bottom right of the structure box. A new window with the MHC 3D structure will open. Then, right click on the 3D image (or ctrl-click for Mac users), then:
a. "Select"-->"all"
b. "Style"-->"structures"-->"backbone"
Now this looks a lot more like the previous MHC model image from parts 1-5.
Click here to reload original:
Surprisingly, the fit of the peptide is not incredibly tight, and many different peptides will fit into any given MHC molecule.
- 7. If you were to alter amino acids in the MHC molecule to (a) affect binding to the peptide, which would you alter? Keep in mind that amino acids within the alpha helices and beta sheets that have side chains that project towards the bound peptide play a crucial role in peptide binding. (b) affect binding to the T cell receptor molecule, which would you alter? (Remember that the T-cell receptor recognizes the whole peptide/binding cleft region as a whole, including amino acids that don’t actually contact the peptide). List 2 amino acids for each answer. (To answer this, move your cursor over an amino acid side chain (light blue) on the image in the area where you would alter something and let the program identify the amino acid for you (three letter code followed by a number at the beginning of the ID string that pops-up).
The following two questions can be answered without the computer. You may want to skip them for now and keep working. Just make sure that you come back to them later!
- 8. What might be the role of the portion of the MHC not involved in direct binding to the peptide? Use your imagination, look at pictures in your book (Chapter 43), or look up and read about MHC on the web (wikipedia would work) and list as many as you can - don’t worry about the true answer, yet!
- 9. A very similar molecule in your immune system is called MHC Class II. (See diagrams in Campbell, Chapter 43 or on wikipedia.) This molecule is structurally and functionally analogous to an MHC Class I molecule. Juvenile diabetes is a disease caused (at least in part) when your MHC Class II molecules erroneously present a peptide from your pancreas. This targets the cells of your pancreas for destruction by your immune system and hence the disease -- called an "autoimmune disease" because you are “immune” to yourself. Interestingly, the cause is a mutation in the genes that code for the MHC molecule. From what you have learned so far, it would be reasonable to assume that the mutation has affected the binding of the MHC to the pancreatic peptide. BUT, while this is in part true, the mutation also disrupts a “salt-bridge” (a weak ionic bond) between the two alpha helices. (An example would be a bridge between Asp77 and His151 in the MHC Class I protein). How could this affect peptide binding? Think back to what you know about how a protein shape is determined.
