Design and Testing of High-Affinity Mutants of Interferon Gamma Receptor 1

Abstract

Specific protein-protein interactions control many crucial processes of the living cell. We aim at elucidating specificity of the interactions at the model system of interferon gamma (IFNg) and its cellular receptor 1 (IFNgRec1), the system important in innate immunity. To modulate (increase as well as decrease) specificity of the interaction we searched for mutations of the receptor molecule that was subjected to in silico mutations using the crystallographically determined structures of IFNg/IFNgRec1 complex. Amino acid substitutions were modeled by empirical force field implemented in the web-based software FoldX. About twenty computer-selected candidate mutants of IFNgRec1 were successfully expressed in Escherichia coli, purified to homogeneity and their affinities to IFNg were determined by surface plasmon resonance (SPR). The SPR measurements showed that affinity of most receptor variants designed for affinity increase had their affinity virtually unchanged, a few had affinity slightly lower but a few bound with affinity significantly higher. Simple and computationally cheap method was therefore able to predict increase of affinity. The receptor variants with increased affinity may be used for diagnostic purposes.Acknowledgements. Support from grant P305/10/2184 from the Czech Science Foundation is greatly acknowledged. All authors are supported by the institutional grant AV0Z50520701.