Ran-binding protein 3 (RanBP3) is a primarily nuclear Ran-binding protein that functions as an accessory factor in the Ran GTPase system. RanBP3 associates with Ran-specific nucleotide exchange factor RCC1 and enhances its catalytic activity towards Ran. RanBP3 also promotes CRM1 -mediated nuclear export as well as CRM1-independent nuclear export of beta-catenin, Smad2, and Smad3. Nuclear import of RanBP3 is dependent on the nuclear import adaptor protein importin-alpha and, RanBP3 is imported more efficiently by importin-alpha 3 than by other members of the importin-alpha family. Protein kinase signaling pathways control nucleocytoplasmic transport through phosphorylation of RanBP3 at Ser58, immediately C-terminal to the nuclear localization signal (NLS) in the N-terminal region of RanBP3. Here we report the crystal structure of human importin-alpha 3 bound to an N-terminal fragment of human RanBP3 containing the NLS sequence that is necessary and sufficient for nuclear import. The structure reveals that RanBP3 binds to importin-alpha 3 residues that are strictly conserved in all seven isoforms of human importin-alpha at the major NLS-binding site, indicating that the region of importin-alpha outside the NLS-binding site, possibly the autoinhibotory importin-beta 1-binding domain, may be the key determinant for the preferential binding of RanBP3 to importin-alpha 3. Computational docking simulation indicates that phosphorylation of RanBP3 at Ser58 could potentially stabilize the association of RanBP3 with importin-alpha through interactions between the phosphate moiety of phospho-Ser58 of RanBP3 and a cluster of basic residues (Arg96 and Lys97 in importin-alpha 3) on armadillo repeat 1 of importin-alpha. (C) 2017 Elsevier Inc. All rights reserved.