Increasing affinity between interferon gamma and its receptor by computer design of receptor mutations. II. Affinity measurements

Pavel Mikulecký, Jiří Černý, Lada Biedermannová, Hana Petroková, Milan Kuchař, Petr Malý, Peter Šebo, and Bohdan Schneider

Institute of Biotechnology AS CR, v. v. i., Vídeňská 1083, CZ-142 20 Prague, Czech Republic

pavel.mikulecky@img.cas.cz, http://www.structbio.eu/BS

Molecules of living systems form a complex maze of interdependent networks and their proper functioning depends on intermolecular interactions. We study protein-protein interactions of cell signaling on a model of human interferon gamma (IFNg) and its cellular receptor (IFNgRec1). In this study, we show successful expression, purification and binding analysis of eighteen mutants of the recombinant IFNgRec1 where mutations were designed by computer modeling (see presentation by Schneider et al.) to increase (14 mutants) and lower (4 mutants) the affinity. The measurements of kinetic (k_on, k_off) and thermodynamic (K_D) constants of the IFNg/IFNgRec1 interaction were performed by surface plasmon resonance. The measured K_D value of recombinant wild type receptor is in agreement with the literature value. Of the selected positive mutants, four have slightly better affinity to IFNg than the wild type receptor and two have their affinity increased significantly. Therefore, our results indicate that rational design of mutations of proteins based on relatively simple and cheap computer models can predict replacement of amino acid residues that change the interaction in the desired direction. Such an approach can lead to creating mutants useful for better understanding of protein-protein interactions and may also have practical use in detection of IFNg.

Acknowledgements. Support from grant P305/10/2184 from the Czech Science Foundation is greatly acknowledged. All authors are supported by institutional grant AV0Z50520701.