We describe a generally applicable computer-based protocol to design protein mutations leading to increase of binding affinity between ligand and its receptor. The method was applied to interferon-γ receptor 1 (IFN-γ-Rx) binding its natural ligand IFN-γ, the system biologically important in innate immunity. We analyzed the four crystallographically independent structures of the IFN-γ-Rx/IFN-γ complex to identify 40 receptor residues forming the interface between the receptor and IFN-γ molecules. For these 40 residues, we performed in silico mutation analysis to select mutations most likely to increase the receptor affinity to IFN-γ by substituting each of the interface ... More
We describe a generally applicable computer-based protocol to design protein mutations leading to increase of binding affinity between ligand and its receptor. The method was applied to interferon-γ receptor 1 (IFN-γ-Rx) binding its natural ligand IFN-γ, the system biologically important in innate immunity. We analyzed the four crystallographically independent structures of the IFN-γ-Rx/IFN-γ complex to identify 40 receptor residues forming the interface between the receptor and IFN-γ molecules. For these 40 residues, we performed in silico mutation analysis to select mutations most likely to increase the receptor affinity to IFN-γ by substituting each of the interface receptor residues by the remaining standard amino acids. The corresponding changes of the free energy were calculated by a protocol consisting of FoldX calculations and molecular dynamics simulations. Based on the obtained values of the free energy changes and on sequence conservation criteria obtained by analysis of 32 receptor sequences from 19 different species we selected 14 receptor variants predicted to strengthen the interaction with IFN-γ.They were all expressed as recombinant proteins in Escherichia coli and their affinities to IFN-γ determined experimentally by surface plasmon resonance, SPR. Of these 14 variants, the simple in silico protocol succeeded in finding two receptor variants with the affinity to IFN-γ increased about five-fold compared tothe wild-type receptor.
