Abstract
Metazoan proteomes contain many protein families wherein paralogs with high sequence and structural similarity have evolved unique functions and binding profiles. We uncovered a region from ciliary protein PCARE is highly specific to Ena/VASP paralog ENAH, but not VASP and EVL (Hwang et al., 2021). Here, we show that despite binding at a site that is identical between paralogs, PCARE stabilizes a conformation of the EVH1 domain of ENAH that is inaccessible to family members VASP and EVL to achieve its high affinity and ∼100-fold specificity. Structure-based modeling rapidly identified seven residues distributed throughout EVL that, when mutated to residues of ENAH, are sufficient to confer high-affinity binding of PCARE. By exploiting the ENAH-specific EVH1 conformation, we rationally designed the tightest and most selective ENAH binder to date, providing a tool for dissecting paralog-specific Ena/VASP functions in processes including cancer cell invasion. Our work uncovers a mechanism of interaction specificity that distinguishes paralogs that share high sequence identity and many common binding partners.
Competing Interest Statement
The authors have declared no competing interest.