Structural and mutational analysis reveals that CTNNBL1 binds NLSs in a manner distinct from that of its closest armadillo-relative, karyopherin α

•The structure of CTNNBL1 includes an abbreviated armadillo domain.•CTNNBL1 is a novel NLS binding protein with a unique carboxy-terminal structure.•Structure-based mutagenesis of CTNNBL1 shows NLS binding distinct from karyopherins. CTNNBL1 is a spliceosome-associated protein that binds nuclear localization signals (NLSs) in splice factors CDC5L and Prp31 as well as the antibody diversifying enzyme AID. Here, crystal structures of human CTNNBL1 reveal a distinct structure from its closest homologue karyopherin-α. CTNNBL1 comprises a HEAT-like domain (including a nuclear export signal), a central armadillo domain, and a coiled-coil C-terminal domain. Structure-guided mutations of the region homologous to the karyopherin-α NLS-binding site fail to disrupt CTNNBL1–NLS interactions. Our results identify CTNNBL1 as a unique selective NLS-binding protein with striking differences from karyopherin-αs. CTNNBL1binds to CDC5L NLS3 by pull down (View interaction) CTNNBL1 and CDC5L NLS3bind by isothermal titration calorimetry (View interaction) CTNNBL1 and Prp31 NLSbind by isothermal titration calorimetry (View interaction)