Begin by downloading the structure of the protein-protein complex. For this, navigate to the protein data bank homepage and enter the PDB ID in the main search box. On the main page for the structure, click on download files, and then on biological assembly 1 to download the files in PDB-gz format.
Open the downloaded structure in UCSF Chimera. Navigate to tools, then structure/editing, and click on change chain IDs. Rename the second chain, initially labeled as A to B.Then, click on favorites, followed by model panel.
Select the model with the two chains, and click on the group/ungroup button to separate each chain into a different model. Next, select the two models and click on the copy/combine button. Enter a new name for the combined model, check close source models, and click okay.
Click on select, then chain, and confirm that the chains in the dimer are now identified as A and B.Click on file, and save PDB to save the edited structure as a new PDB file. For identifying the target segment in the ligand protein, navigate to the BUDE Alanine Scan server. Click on the choose file button under structure, upload, and upload the saved PDB file.
On the next page, check that the structure was correctly loaded and enter a name for the job in the server. Set chains A as a receptor, and B as the ligand, and click on the start scan button to submit the job. Once the job is finished, click on show results to open the results page.
From the residue list, select the stretch of residues predicted to better interact with the target binding surface. Using this protocol, an amino acid segment interacting with IRF5 binding surface was predicted. Using computational alanine scanning mutagenesis, a 13 amino acid segment was predicted from positions 424 to 436, with the PLXIS motif starting at arginine 432.
