Generation of specific inhibitors of SUMO1- and SUMO2/3-mediated protein-protein interactions using Affimer (Adhiron) technology

Abstract

Because protein-protein interactions underpin most biological processes, developing tools that target them to understand their function or to inform the development of therapeutics, is an important task. SUMOylation is the posttranslational covalent attachment of proteins in the SUMO family (SUMO1, SUMO2, or SUMO3) and regulates numerous cellular pathways. SUMOylated proteins are recognized by proteins with SUMO-interaction motifs (SIMs) that facilitate non-covalent interactions with SUMO. Here, we describe the use of the Affimer system of peptide display for rapid isolation of synthetic binding proteins that inhibit SUMO-dependent protein-protein interactions mediated by SIMs both in vitro and in cells. Crucially, these synthetic proteins did not prevent SUMO conjugation either in vitro or in cell-based systems, enabling the specific analysis of SUMO-mediated protein-protein interactions. Furthermore, through structural analysis and molecular modelling, we explored the molecular mechanisms that may underlie their specificity in interfering with either SUMO1-mediated interactions or interactions mediated by either SUMO2 or SUMO3. Not only will these reagents enable investigation of the biological roles of SUMOylation, the Affimer technology used to generate these synthetic binding proteins could be exploited to design or validate reagents or therapeutics that target other protein-protein interactions.