De novo design of modular protein oligomers to investigate cell signaling

Abstract

Clustering of receptors is a critical step in activation of signaling, but this phenomenon is difficult to investigate with native soluble ligands that bind only 1-2 copies of a receptor. I designed a series of modular protein scaffolds with 4-, 6-, and 8-fold symmetry, offering up to 8 sites for display of a receptor-binding domain. Scaffolds were extended by adding repeat units to vary the spacing of the bound receptors. To target receptors, a de novo miniprotein binder to the fibroblast growth factor receptor (FGFR) was attached to scaffolds through genetic fusion. Treatment of cells with the designed scaffolds resulted in colocalization and decreased membrane diffusion of receptors, increased Erk phosphorylation, and intracellular calcium release indicating activation of FGF signaling. These designed protein scaffolds can be applied as a universal tool to a variety of systems to dissect the role of clustering in cell surface receptor-mediated signaling pathways.