Increasing Affinity of Interferon-γReceptor 1 to Interferon-γby Computer-Aided Design

Abstract

We describe a computer-based protocol to design protein mutations increasing binding affinity between ligand and its receptor. The method was applied to mutate interferon-γ receptor 1 (IFN-γ-Rx) to increase its affinity to natural ligand IFN-γ, protein important for innate immunity. We analyzed all four available crystal structures of the IFN-γ-Rx/IFN-γ complex to identify 40 receptor residues forming the interface with IFN-γ. For these 40 residues, we performed computational mutation analysis 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 and molecular dynamics calculations. Based on the computed changes of the free energy and on sequence conservation criteria obtained by the analysis of 32 receptor sequences from 19 different species, we selected 14 receptor variants predicted to increase the receptor affinity to IFN-γ. These variants were expressed as recombinant proteins in Escherichia coli, and their affinities to IFN-γ were determined experimentally by surface plasmon resonance (SPR). The SPR measurements showed that the simple computational protocol succeeded in finding two receptor variants with affinity to IFN-γ increased about fivefold compared to the wild-type receptor.

QTY Code-designed Water-soluble Fc-fusion Cytokine Receptors Bind to their Respective Ligands

“…IFNγR1 QTY -Fc versus IFNγ 1.7 (cell) (Celada et al, 1985); 30.8 (SPR) (Mikulecky et al, 2013) 6.2 AE 3.0…”

Deciphering critical amino acid residues to modify and enhance the binding affinity of ankyrin scaffold specific to capsid protein of human immunodeficiency virus type 1

“…9 Several reports have described successful improvement of the binding affinity of interacting proteins using computationally guided molecular design. [20][21][22][23][24] Computational design is perfectly adapted to this particular goal, since individual amino acid residue(s), or limited protein domain(s), could be targeted and re-designed while maintaining the folding and three -dimensional structure of the whole molecule and its biological properties. 25 As applied to our antiviral scaffold Ank GAG 1D4, a binder of the HIV-1 CA protein, 13,14 the logical start-point of our affinity enhancement strategy was the modification of amino acid residues which were crucial determinants of the interaction of Ank GAG 1D4 with the CA protein.…”

Redesigning Protein Cavities as a Strategy for Increasing Affinity in Protein-Protein Interaction: Interferon-γReceptor 1 as a Model

“…We followed the protocols from our previous study [ 21 ] for all proteins produced in this study. All selected IFN γ R1 variants were prepared, expressed, and successfully purified to homogeneity by the following protocol.…”

“…Melting temperatures of the receptor variants were measured using fluorescence-based thermal shift assay and for selected mutants by CD melting experiments. Interactions between IFN γ R1 variants and IFN γ SC were measured by the technique of surface plasmon resonance (SPR) as discussed in our previous study [ 21 ]. Experimental procedures are detailed below.…”

“…This study follows our previous article [ 21 ] in which we designed mutations increasing the affinity of human interferon- γ receptor 1 (IFN γ R1) towards its natural cognate molecule interferon- γ (IFN γ ), an important protein of innate immunity [ 22 , 23 ]. Here, we retain this model system and the main contours of the protocol but replace the search for interface mutations by searching for mutations in the receptor cavities in order to further increase its interaction affinity to IFN γ and our computer analysis revealed four such cavity mutants.…”