2.3 Hotspot identification using residue scanning
The Ala scan was performed on the prepared library of protein-protein and protein-peptide complexes using the Residue Scanning module in Bioluminate [38]. Ala mutation was carried out, one residue at a time, for all the interface residues. The method calculates relative binding affinity values (Gbind or simply G; also referred as Affinity in the reference [38]) and stability (∆∆Gstability) between the mutant and the wild type protein using implicit solvation based MM-GBSA and using the thermodynamic cycle approach. The details of thermodynamic cycle, which allows one to calculate the net Gbind and ∆∆Gstability, taking the advantage of state function nature of the free energy is described in literature [37,42]. For interface residues, default 4Å distance between residues in either of the protein chains was used [38].
The Δ∆Gbind is the change in binding affinity between binding partners upon point mutation. One of the difference (Δ) is between the bound and unbound state of binding partners using MM-GBSA and the another difference (Δ) is between wild type and the mutant. The positive value indicates loss in binding affinity upon mutation to Ala and negative value indicates gain in affinity. The calculations were carried out using the Schrödinger Prime MM-GBSA, which uses an implicit (continuum) solvation model [43]. ΔGstability or ΔStability (solvated) is the change in the stability of the protein upon mutation, also calculated using the Prime energy function. The stability was defined as the difference in free energy between the folded state of molecule and the corresponding unfolded state were estimated from Gly-X-Gly tripeptide, where X is the residue under consideration [38]. A negative value refers that the mutant is more stable. Residue involving covalent linkage (having Δ∆Gbind> 80 kcal/mol) were discarded. Finally, all the hotspot residues with Δ∆Gbind in 2-80 kcal/mol range were considered for analysis. All the results were analysed using Maestro visualizer, Bioluminate utilities and Microsoft Excel.