Ubiquitin-derived artificial binding proteins targeting oncofetal fibronectin reveal scaffold plasticity by β-strand slippage

Abstract

AbstractAffilin proteins (artificial binding proteins based on the ubiquitin scaffold) were generated using directed protein evolution to yieldde-novovariants that bind the extra-domain B (EDB) of oncofetal fibronectin, an abundant tumor marker in fetal and neoplastic tissues. Structures of two EDB-specific Affilin molecules reveal striking structural plasticity of the ubiquitin scaffold, characterized by β-strand slippage, leading to diverse register shifts of the β5 strands. This recruits residues from β5 to a loop region, enhancing the target-binding interface. The observed β-strand rearrangements, manifested by pressure of selection for target binding, challenge the accepted paradigm that directed evolution of binding proteins should base on rigid frameworks. Fold plasticity allowing β-strand slippages enhances the evolutionary potential of proteins beyond “simple” mutations significantly and provides a general mechanism to generate residue insertions/deletions in proteins. They are however difficult to predict, underlining the need for caution in interpretation of structure-activity relationships of evolved proteins.