To provide examples on how the web server can be used for protein binding site prediction, we illustrate some case studies used to analyse protein-drug interactions in different perspectives.
Uncharacterized protein is a protein of which the function cannot be defined, either based on sequence or structural similarity search. Meanwhile, hypothetical protein is a protein whose existence in the genome has been predicted, but the expression cannot be proved via experimental validation. While various sequence and structure- based prediction tools enable the prediction of function for these proteins, the prediction of drug binding site or the ability of the protein to bind to drug molecules is one of the potential method used for functional annotation.
One example of hypothetical protein is an uncharacterized protein from Parabacteroides merdae (PDB ID: 4r7f) obtained from the Joint Center for Structural Genomics (JCSG). From the ‘Search for amino acid arrangements similar to known drug binding sites in known target structures / protein-drug complexes’ interface, PDB ID search for ‘4r7f’ returns structural details and visualization of the protein structure, as well as a list of similar patterns of amino acids derived from ASSAM searches. User may filter the list based on RMSD, Z-score and sequence identity values, or select only matches from human structures only.
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User can click on the DrReposER ID to get more details related to known drug binding sites having similar patterns of amino acids as the query structure.
Clicking the 'View' link on the last column will shows superposed patterns of amino acids in the NGL viewer.
If the search return no matches, user may use the 'Search a protein structure for amino acid residue arrangements similar to a known drug binding site' interface, where user can upload a PDB-formatted structure or insert a PDB ID to search for potential motifs for drug binding based on SPRITE search. SPRITE search compares a query protein structures against a data set of known drug binding sites and yield matches of amino acid patterns.
PDB ID 5gkx mapped to a crystal structure of highly conserved hypothetical protein, TON_0340 from Thermococcus onnurineus. The protein is found to be conserved among different organisms including human and not homologous to any characterized proteins. It is proposed to be a Manganese-Dependent Phosphatase due to the discovery of metal binding site and ability of this protein to bind to manganese ions.
A query for '5gkx' returns list of similar patterns for right-handed and left-handed superposition of amino acid patterns.
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User may select to view either the list for left-handed or right-handed superpositions of amino acid patterns. Clicking on any list of hits will open a new page with an NGL viewer showing the crystal structure of the protein and a list of similar patterns of amino acids found from SPRITE search.
User may filter the results according to residue number or RMSD value. Clicking on the DrReposER ID on the second column will open a new page for selected DrReposER ID and details for match binding site.
User may view one or more potential motifs for the query protein by selecting the pattern on the first column. Selecting only one pattern will give a better view in teh NGL viewer.
The NGL viewer provides multiple options for user to select, either by showing the superposition of matched residues or showing the actual known binding sites.
Clicking on the 'Superposed motifs' button will shows only matched residues that are superposed to the residues from query protein.
Clicking on the 'Hit binding sites' button will shows the actual known binding site that match the query structure.
Predicted binding sites obtained from structural similarity searches can be further used for molecular docking analysis and further validation through experiments.
References:
1) Jin, M. et al. (2013). Discovery of potent, selective and orally bioavailable imidazo[1,5-a]pyrazine derived ACK1 inhibitors. Bioorganic & Medicinal Chemistry Letters. 23(4):979-984. (link)
2) Mahajan, K. & Mahajan, N. P. (2013). ACK1 Tyrosine Kinase: Targeted Inhibition to Block Cancer Cell Proliferation. Cancer Lett. 338(2):185-192. (link)
3) Sohn et al. (2016). Identification of a Highly Conserved Hypothetical Protein TON_0340 as a Probable Manganese-Dependent Phosphatase. PLoS ONE 11(12): e0167549. (link)