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

Combining computational and experimental tools, we present a new strategy for designing high affinity variants of a binding protein. The affinity is increased by mutating residues not at the interface, but at positions lining internal cavities of one of the interacting molecules. Filling the cavities lowers flexibility of the binding protein, possibly reducing entropic penalty of binding. The approach was tested using the interferon-gamma receptor 1 (IFN gamma R1) complex with IFN gamma as a model. Mutations were selected from 52 amino acid positions lining the IFN gamma R1 internal cavities by using a protocol based on FoldX prediction of free energy changes. The final four mutations filling the IFN gamma R1 cavities and potentially improving the affinity to IFN gamma were expressed, purified, and refolded, and their affinity towards IFN gamma was measured by SPR. While individual cavity mutations yielded receptor constructs exhibiting only slight increase of affinity compared to WT, combinations of these mutations with previously characterized variant N96W led to a significant sevenfold increase. The affinity increase in the high affinity receptor variant N96W+V35L is linked to the restriction of its molecular fluctuations in the unbound state. The results demonstrate that mutating cavity residues is a viable strategy for designing protein variants with increased affinity.