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.