Evolutionary diversification of PDZ domains drives substrate specificity switching in LNX E3 ubiquitin ligases

How paralogous proteins acquire distinct substrate specificities remains a central question in molecular evolution. We reveal an evolutionary switch in substrate targeting between LNX paralogs, E3 ubiquitin ligases that mediate proteasome-dependent degradation of the cell fate determinant NUMB. In teleost fish and amphibians, Numb degradation is primarily mediated by Lnx2, whereas Lnx1 exhibits minimal activity toward Numb. In contrast, in amniotes, this specificity is reversed, with LNX1—rather than LNX2—mediating Numb degradation, revealing a lineage-specific functional switch. Mechanistically, we show that Numb targeting by human LNX1 is governed by the cooperative action of its PDZ domains, particularly PDZ2 and PDZ3. Domain swapping experiments demonstrate that replacement of human PDZ domains with zebrafish counterparts abolishes Numb degradation, whereas reciprocal introduction of human PDZ domains into zebrafish Lnx1 confers robust Numb-targeting activity. Phylogenetic analysis further captures an evolutionary “snapshot” in amphibians, where progressive sequence divergence within PDZ domains precedes the functional transition observed in amniotes. Together, our findings demonstrate that modular evolution of PDZ domains drives a rewiring of substrate specificity between LNX paralogs, providing a mechanistic framework for how gene duplication enables functional diversification of protein interaction networks.