Exercise: Modeling of epitopes

B-cell epitope prediction

We want to look for epitopes on the HIV GP120 protein. We will use both sequence-based and structure-based methods.

Input data.

Go to the pdb web-page and look for the pdb entry 4LSP. It is GP120 complexed with a broadly neutralizing antibody.

Q1: Download both the structure (pdb) and the sequence (fasta) of the proteins in this entry. Which proteins are in chain G, L and H respectively?

Q2: using pymol, can you identify which residues are part of the epitope? Is the epitope linear? Does it have a linear core?

Hints: remove the waters, then select the antibody and modify this selection to find residues in a 6Å sphere.

Q3: using pymol, identify the antibody in the structure, select and remove it (action -> remove atoms). Save the resulting molecule as antigen.pdb

Now we are starting with a sequence-based prediction of the epitopes.

Bepipred

Go to the bepipred website and upload the sequence of GP120. Leave the default parameters and submit the job. Don’t close the window because we will need it again!

Q4: does the Bepipred prediction overlap with the actual epitope?

Be careful: pdb file numbering does not always start from 1! Compare the numbering of the pdb with the one returned by Bepipred.

BEpro

Now let’s try a structure-based method: BEpro Go to the website and submit the antigen.pdb file to the server. Select the “PDB File” output. Submit the job and save the results as bepro.pdb

The prediction is “hidden” in the b-factor field of the pdb (this is a standard way to add an annotation to a structure)

Load the prediction in pymol, then color it (only it, not the other molecules!) using spectrum->b-factor

Q5: Does the prediction overlap with our epitope?

Discotope 2.0

Finally, let’s use the CBS Discotope server.
Load the antigen.pdb file and submit the prediction. Save the output as discotope.pdb
Once again, the prediction is in the b-factor field (but you can download it in other formats as well)
Load it in pymol and colour according to the b-factor.

Q6: Does the prediction overlap with our epitope?

Q7: Which prediction best matches our epitope?

Q8: Use Discotope to predict the epitopes in the simian HIV GP120 homolog (pdb code 2BF1). Compare the results. Try and explain the differences. According to you is it a meaningful result or not?