title: Increasing affinity of interferon-γ receptor 1 to interferon-γ by 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.
authors.affiliation: Institute of Biotechnology AS CR, v. v. i., Vídeňská 1083, 142 20 Prague, Czech Republic.
authors.forename: Pavel
authors.initials: P
authors.lastname: Mikulecký
authors.forename: Jiří
authors.initials: J
authors.lastname: Cerný
authors.forename: Lada
authors.initials: L
authors.lastname: Biedermannová
authors.forename: Hana
authors.initials: H
authors.lastname: Petroková
authors.forename: Milan
authors.initials: M
authors.lastname: Kuchař
authors.forename: Jiří
authors.initials: J
authors.lastname: Vondrášek
authors.forename: Petr
authors.initials: P
authors.lastname: Malý
authors.forename: Peter
authors.initials: P
authors.lastname: Šebo
authors.forename: Bohdan
authors.initials: B
authors.lastname: Schneider
mesh_terms: D019943:Amino Acid Substitution; D006801:Humans; D007371:Interferon-gamma / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D056004:Molecular Dynamics Simulation; D017510:Protein Folding; D017471:Receptors, Interferon / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D020349:Surface Plasmon Resonance; D000096743:Interferon gamma Receptor
chemical_list: D007371:Interferon-gamma; D017471:Receptors, Interferon; D000096743:Interferon gamma Receptor; C573415:IFNG protein, human