Ghosh et al. , showed that by applying the random alanine scanning mutation, it was possible to validate how much the residues from the BB-and DD-loops of the TIR domain contribute to TLR2 heterodimer complex formation. For that, the binding free energy (ΔΔGbinding) of the interface residues was computed. The residues with positive cut-off values > 0.5 kcal/mol were accepted as the residues of importance in the dimer stability for human TLR1-2 and TLR2-6.