Bcell Epitope prediction
B-cell epitope prediction — Why do some flu vaccines miss?
Influenza A hemagglutinin (HA) has two very different antibody targets: the variable head and the conserved stem. Head-binding antibodies often lose potency as the virus drifts; stem-binding broadly neutralizing antibodies (bnAbs) can resist drift. Can standard sequence-based and structure-based B-cell epitope predictors tell these stories apart?
Go to the pdb homepage and look for the pdb entries 4FQI and 7K39. It is hemagglutinin protein from Inlfuenza complexed with two different antibodies.
Q1: Download both structures (pdb) and open them in separate PyMOL windows. One of them is a tri-merthe sequence (fasta) of the proteins in this entry. Which are the chains from HA, and the antibody (L and H) respectively?
hint: you can find this information in the structure web-page, in the PDB file itself (you can open it with a text editor and search for COMPND)
COMPND MOL_ID: 1; COMPND 2 MOLECULE: HEMAGGLUTININ HA1; COMPND 3 CHAIN: A; COMPND 4 FRAGMENT: UNP RESIDUES 17-346; COMPND 5 SYNONYM: HEMAGGLUTININ RECEPTOR BINDING SUBUNIT HA1; COMPND 6 ENGINEERED: YES; COMPND 7 MOL_ID: 2; COMPND 8 MOLECULE: HEMAGGLUTININ HA2; COMPND 9 CHAIN: B; COMPND 10 FRAGMENT: UNP RESIDUES 347-520; COMPND 11 SYNONYM: HEMAGGLUTININ MEMBRANE FUSION SUBUNIT HA2; COMPND 12 ENGINEERED: YES; COMPND 13 MOL_ID: 3; COMPND 14 MOLECULE: ANTIBODY CR9114 HEAVY CHAIN; COMPND 15 CHAIN: H; COMPND 16 FRAGMENT: FAB; COMPND 17 ENGINEERED: YES; COMPND 18 MOL_ID: 4; COMPND 19 MOLECULE: ANTIBODY CR9114 LIGHT CHAIN; COMPND 20 CHAIN: L; COMPND 21 FRAGMENT: FAB LAMBDA; COMPND 22 ENGINEERED: YES
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 5Å sphere. You can find a quick video on how to do this in the hands-on prerequisites.
Q3: using pymol, identify the antibody in the structure, select and remove it (action -> remove atoms). Save the resulting molecule as antigen.pdb
You can save using the GUI (remember to select PDB as option) as well as typing "save antigen.pdb" in the command line
Now we are starting with a sequence-based prediction of the epitopes.
Bepipred 2/3
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.
Repeat the prediction now using BepiPred3.
Q5: Does the prediction overlap with our epitope now?
Q6: What is different in BepiPred 2 compared to Bepipred 3? Discotope 2/3
Finally, let's use DTU 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.
Q7: Does the prediction overlap with our epitope?
Q8: Which prediction best matches our epitope?
Q9: Use Discotope to predict the epitopes in the simian HIV GP120 homolog (pdb code 3FUS). If you want to save time, use this pre-cleaned PDB. Compare the results. Try and explain the differences. According to you is it a meaningful result or not? Please note that some bugs might occur, according to which file you use. If some strange results appear, report it in your answer :)
Q10: Compare the results obtained in Discotope 2 and 3