A detailed explanation of results obtained using "1CRN.pdb":http://www.pdb.org/pdb/explore.do?structureId=1CRN as input file in HORI server is explained in Section - 12 of PROCEDURE. HORI server can be utilized to understand interesting higher-order interactions from protein structures. Detailed analysis of different higher order interactions and residues that mediate such interactions using set of protein structures of interest will be providing better insights to understand structural features.
Application of HORI server in protein structure analysis:
Users can utilize the different programs for calculating higher order residue interactions in protein structures in different contexts. For example the higher order interactions can be utilized in the identification of alternate binding sites based on higher order residue interactions, interaction patterns for mutational studies and protein engineering, analysis of higher order residue interactions of active site residues, to distinguish protein folds through map of higher order interactions. As a general protein structure analysis web server that can perform computation of higher order residue interactions from a single residue level to whole structure. In order to illustrate the usefulness of higher order interactions and to demonstrate where triplet and quadruplet interactions might be crucial, we take the example of four different proteins belong to different superfamily from SCOP database 23, 24.
Example 1: PDB ID: 1NNQ
3D Domain swapping is a structural phenomenon observed in proteins with diverse sequence, structure and function25-27. Ruberythrin from Pyrococcus furiosus is reported to be involved in 3D domain swapping28. We have used the structure (PDB ID: "1NNQ":http://www.pdb.org/pdb/explore.do?structureId=1NNQ) and performed a HORI-Global based computation and retrieved all pair wise interactions. HORI server reported a total of 580 pair wise interactions with in the cut-off of 1Å to 7Å. Out of these pairwise interactions, 13% of interactions are mediated by the residues between the two chains. Such detailed analysis on proteins with peculiar structural features will give a better understanding of the role higher order interactions in orchestrating the structural integrity.
Example 2: PDB ID: 1BF2
Isoamylase from Pseudomonas amyloderamosa (PDB ID: "1BF2":http://www.pdb.org/pdb/explore.do?structureId=1BF2) 29 is a hydrolase reported with active site residues ASP, GLU, ASP. We have used these active site residues reported in Catalytic Site Atlas30 database and calculated the triplet interactions mediated by this active site residues. The link to access the results is provided in Table 1.
Example 3: PDB ID: 1QJ4
Hydroxynitrile lyase is a lyase from Hevea brasiliensis (PDB ID: "1QJ4":http://www.pdb.org/pdb/explore.do?structureId=1QJ4) with the classical Ser, His, Asp triad. The active sites are reported in Catalytic Site Atlas based on PSI-BLAST alignment to the PDB ID: 1C4X. We performed the option to compute quadruple distances around any 3 residues. List of quadruple interactions mediated by active site is obtained. This kind of analysis will be useful to identify potential alternate active site and residues suitable for mutational experiments. Results can be accessed from the link provided in Table 1.
Example 4: PDB ID: 1AQ0
The functionally important residues, including at the active site, may not be strictly conserved for protein domains at the level of superfamily31. HORI server can be used to search for alternate active site residues based on specific higher order residue interactions. Barley 1,3-1,4-beta-glucanase32 from Hordeum vulgare belong to the TIM barrel glycosyl hydrolases superfamily. This PDB entry (PDB ID: "1AQ0":http://www.pdb.org/pdb/explore.do?structureId=1AQ0) belongs to the Glycosyl hydrolases family 17 (Pfam ID: "PF00332":http://pfam.sanger.ac.uk/family/PF00332) in Pfam database33, 34. We have used three of the four active site residues reported in Catalytic Site Atlas30 in the option to compute quadruple distances around any three residues in a PDB file and successfully identified the fourth active site residues in a quadruple interaction flexibly.
Links to access HORI-Global results or automated example pages to run the computation of higher order residues interactions mentioned in this protocol is provided in Table 1.
Currently the higher order residue interaction calculation is only possible through the web interface available from the URL: "http://caps.ncbs.res.in/hori/hori.html":http://caps.ncbs.res.in/hori/hori.html. We will be extending this platform for the programmatic access through a web service implementation of the programs using "REST":http://en.wikipedia.org/wiki/Representational_State_Transfer and "SOAP":http://en.wikipedia.org/wiki/SOAP based technologies.
Scope of protocol based on HORI Server:
Higher order interactions calculated using set of computationally intensive algorithms available in HORI server will be useful in protein structure prediction, protein modelling, protein-protein interaction, active site identification and to understand higher order interaction characteristics of active site residues within specified distance shells. Knowledge about the higher order interaction will be of great importance in the structural biology domain due to its wide range of applications in fold recognition, structural analysis, protein engineering and to understand mechanism of action of enzymes.