Evolutionary analysis through structural modeling of FAM222 proteins reveals a novel conserved disordered domain in vertebrates that interacts with NLK

Intrinsically disordered regions (IDRs) play essential roles in signaling by mediating dynamic, yet specific protein-protein interactions. Many IDRs have evolved as kinase-binding modules, particularly in mitogen-activated protein kinase (MAPK) networks, where they facilitate critical regulatory interactions for signaling specificity. However, the evolutionary constraints that shape IDRs and their role in MAPK regulation remain poorly understood. Here, we identify DCD222, a highly conserved IDR within the FAM222 protein family, which interacts with Nemo-like kinase (NLK), an atypical MAPK involved in WNT, NOTCH, VEGF, mTOR and TGF-β signaling. Phylogenetic analysis reveal that DCD222 has been conserved from cyclostomes to humans, indicating strong selective pressure on its function. Using AlphaFold3, we show that DCD222 binds to NLK through an extended mechanism, where a reverse docking (revD) motif sequence engages the NLK docking groove in the C-lobe, while a β-hairpin-like and β-sheet motif are inserted into the N-lobe. The interaction does not obstruct the kinase’s catalytic site, suggesting that the proteins FAM222A and FAM222B are a potential modulator of NLK rather than a simple substrate. Our findings establish DCD222 as an evolutionarily constrained IDR, providing new information into how disordered domains contribute to signaling adaptation in vertebrates.