Examination of protein-ligand interactions through bioinformatics and its relation to drug design
Background:
Proteins bind ligands/substrates through molecular interactions provided by specific amino acids in the binding pocket. These interactions (e.g. hydrogen bond, hydrophobic interactions, and electrostatic interactions etc.) are key to binding and in turn modulating the proteins function. As a result, one common drug design strategy is to design molecules that would make favorable interactions to these essential amino acids in order to outcompete the native substrate and inhibit the proteinâ€™s function. At the same time, regions that can change can be important to take advantage of for designing organism specific drugs and avoiding off-target effects (e.g. maybe you want the drug inert in humans but highly functional in dogs). In this case you want to take advantage of the sequence differences between species. In this lab, you are going to identify and visualize these important amino acids through protein sequence analysis using bioinformatics tools.
Instructions:
1. We are going to use a sequence visualization program (Jalview). This is where we can identify the residues we think are interacting with the molecule in the active site.
2. On the page of your protein from the pdb website, click on fasta sequence under the â€œDisplay Filesâ€ menu.
3. This will display the fasta sequence on your browser (which is the amino acid sequence for your enzyme).
4. Copy this sequence then go to the BLAST web server
5. Select Protein Blast (blastp)
6. Paste in your sequence. Wait! Donâ€™t hit submit, instead lets add some paramters to make sure you have enough diversity in sequences to get a good analysis of conservation:
a. Change database to: Reference Proteins (refseq_protein). This database is a bit less redundant than the default nr database. Ideally we would use a database that is nonredundant on the 75% level (other servers such as HMMER allow that, but they are more difficult to use).
b. Expand â€œAlgorithm Parametersâ€ and select 250 on â€œMax Target Sequencesâ€. This will
make sure you have enough sequences to measure conservation while making sure that all the proteins identified are predicted to be highly related.
*IMPORTANT NOTE: If you see %â€™s in the â€œIdentâ€ column in the blast results going below 30% re-adjust this to 100 or even 50. Below 30% you are in what is considered the â€œtwighlightâ€ zone and the sequences you are finding are not necessarily related in structure or function. Basically, you have drifted too far in evolutionary space. Roughly speaking, 30-70% Ident it has been found that proteins are both related in protein structure and function, above 70% it is almost certain that proteins are almost identical in structure and function.
7. Hit â€œBLASTâ€ to initiate search results
8. Analyze your results, make sure all the hits are â€œPink or Redâ€ for alignment score (i.e. large portions of the sequence aligned). And in the â€œDescriptionsâ€ table with â€œSequences producing significant alignmentsâ€ make sure the Ident column never goes below 30. If it does, see note above.
9. If all looks good, Select â€œMultiple Alignmentâ€ near the top in the â€œOther Reportsâ€ section.
10. From this output (donâ€™t worry if an error is thrown for graphical overview) select:
â€œDownloadâ€ -> Fasta plus gaps
13. Now we are going to open another program named Jalview (http://www.jalview.org/). Click Launch Jalview Desktop open the downloaded file.
14. Under the â€œFileâ€ menu, hit Input Alignment, from file and open the alignment file you just downloaded from the BLAST.
15. Under Jalview hit colour tab at top, then click percentage identity, now go back to color and click above identity threshold. Change occurrence to 99% conserved.
15. The residues highlighted here are either critical for protein structure or function. All of the residues you identified in lab 1 (i.e. the catalytic residues) should be highlighted.
16. Using Jalview and using percentage identity and moving the bar back and forth you can determine the percent conservation for a residue (look at switch from being
colored to not colored).
17. Now, in the assignment below you will need to find non-catalytic residues (i.e. residues not directly involved in the reaction mechanism) that are either highly conserved OR have low conservation. Roughly speaking (based on search paramters above):
High Conservation residues have >70% identity across the homologous sequences
Low Conservation residues have <30% identity across the homologous sequences
Residues in the 30-70% range a likely important but not critical for structure or function.
Assignment for lab 2
For your assigned enzyme group complete the following assignments with the help of the Lab 2 instructions:
1) What NON-CATALYTIC residues in the active site of the enzyme are most likely important for function?
a. Highlight three highly conservative residues, show both structural interactions AND conservation using PyMol and JalView. Hypothesize why they are critical for function.
b. Highlight one low conservation residue, show both structural interactions AND conservation using Pymol and JalView. What other amino acids are observed, hypothesize why the alternate residues are ok.
2) How does the inhibitor interact and how would you further optimize?
a. Describe WHY it acts as an inhibitor.
b. What key interactions are made with the inhibitor? Do any of those interactions mimic the natural substrate?
c. If you were to modify the inhibitor what new interactions would you try to take advantage of? (hintâ€¦conserved residues in the protein are unlikely to change while maintaining protein structure or function)
Case 1 = ACE: 2X91
Case 2 = OD: 2TOD
Case 3 = Î±-Fucosidase: 2ZX5
Case 4 = PNP: 1A9S
Case 5 =CD: 2FR6

Examination of protein-ligand interactions through bioinformatics a...