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

Abstract

Affilin proteins (artificial binding proteins based on the ubiquitin scaffold) were generated using directed protein evolution to yield de‐novo variants 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.