Crystal structure of importin-α3 bound to the nuclear localization signal of Ran-binding protein 3

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Highlights

  • Ran-binding protein 3 (RanBP3) is an accessory factor in the Ran GTPase system.

  • The N-terminal region of RanBP3 contains a nuclear localization signal (NLS).

  • Crystal structure of importin-α3 bound to the NLS of RanBP3 is solved.

  • Phosphorylation of RanBP3 at Ser58 may modulate binding affinity to importin-α.

Abstract

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 β-catenin, Smad2, and Smad3. Nuclear import of RanBP3 is dependent on the nuclear import adaptor protein importin-α and, RanBP3 is imported more efficiently by importin-α3 than by other members of the importin-α 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-α3 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-α3 residues that are strictly conserved in all seven isoforms of human importin-α at the major NLS-binding site, indicating that the region of importin-α outside the NLS-binding site, possibly the autoinhibotory importin-β1-binding domain, may be the key determinant for the preferential binding of RanBP3 to importin-α3. Computational docking simulation indicates that phosphorylation of RanBP3 at Ser58 could potentially stabilize the association of RanBP3 with importin-α through interactions between the phosphate moiety of phospho-Ser58 of RanBP3 and a cluster of basic residues (Arg96 and Lys97 in importin-α3) on armadillo repeat 1 of importin-α.

Introduction

Ran is a Ras-related small GTPase that is involved in diverse cellular processes, including nucleocytoplasmic transport, mitotic spindle assembly, and post-mitotic nuclear assembly (reviewed in Ref. [1]). Ran-binding protein 3 (RanBP3) is an accessory factor in the Ran GTPase system and belongs to a family of proteins that share a homologous Ran-GTP binding domain (RanBD) of about 120 amino acids [2]. N-terminal to the RanBD, RanBP3 has phenylalanine-glycine (FG)-repeat motifs, characteristic of a subgroup of nucleoporins localized at nuclear pore complexes [2]. Unlike nucleoporins, however, RanBP3 shows a diffuse intranuclear distribution excluding nucleoli in interphase cells [2]. RanBP3 associates with Ran-specific nucleotide exchange factor RCC1 and enhances its catalytic activity towards Ran [3]. RanBP3 accelerates formation of the CRM1 nuclear export complex [4] and promotes CRM1-mediated nuclear export [5], [6]. RanBP3 also facilitates CRM1-independent nuclear export of β-catenin, Smad2, and Smad3, thereby negatively regulating Wnt signaling and TGF-β signaling [7], [8].

Nuclear import of RanBP3 occurs via importin-α (Impα)-importin-β1 (Impβ1)-dependent pathway [9]. In this classical nuclear import pathway, the Impα adaptor proteins bind cargo proteins possessing the nuclear localization signal (NLS), and heterodimerize with Impβ1 through the N-terminal Impβ1-binding (IBB) domain, forming nuclear import complexes that carry cargo proteins from the cytoplasm to the nucleus through nuclear pore complexes (reviewed in Refs. [10], [11]). The IBB domain of Impα has NLS-like sequence that inhibits the binding of NLS-containing cargo to the NLS-binding armadillo (ARM) repeat domain of Impα [12]. The association of Impβ1 with the IBB domain of Impα relieves the autoinhibition by the IBB domain, and thereby increases the affinity of NLS-cargo to Impα [13]. Human cells have seven Impα isoforms (Impα1, Impα3, Impα4, Impα5, Impα6, Impα7, and Impα8), each of which has different substrate specificity (reviewed in Refs. [14], [15]). It has been shown that RanBP3 is imported more efficiently by Impα3 than by other members of the Impα family [9].

The residues 40–57 of RanBP3 (the residue number refers to that of isoform 3, also known as RanBP3-b, which appears to be the primary transcript [9]) have been identified as the NLS sequence that is necessary and sufficient for nuclear import [9]. Welch et al. suggested that this is an “unusual” NLS that binds preferentially to Impα3 [9]. Interestingly, protein kinase signaling pathways (the PI3K/Akt and Ras/ERK/RSK pathways) control nucleocytoplasmic transport through phosphorylation of RanBP3 at Ser58, immediately C-terminal to the NLS, by unknown mechanisms [16], [17], [18], [19]. In the present study, we report structural characterization of the interactions between the RanBP3 NLS and Impα3.

Section snippets

Preparation of ΔIBB Impα3-RanBP3 NLS complex for crystallization

N-terminally His6-and S-tagged ΔIBB Impα3 (human, residues 70–485; UniProt code, O00629) and N-terminally GST-tagged RanBP3 NLS (human, isoform 3, also known as RanBP3-b, residues 31–60; UniProt code, Q9H6Z4) were expressed separately from pET30a-TEV [20] and pGEX-TEV [20], respectively, in the E. coli host strain BL21-CodonPlus(DE3)RIL (Stratagene). After harvesting, the two sets of cells were mixed, suspended in buffer A [30 mM Tris-HCl pH 7.5, 10 mM imidazole, 500 mM NaCl, 1 mM

Crystal structure of Impα3 bound to non-phosphorylated NLS of RanBP3

To elucidate the mechanism of RanBP3 NLS recognition by Impα3, we grew crystals and solved the 3.0 Å-resolution structure of the NLS-binding ARM repeat domain of human Impα3 bound to human RanBP3 (residues 31–60, non-phosphorylated) (Fig. 1). The structure was refined to free and working R-factor values of 28.1% and 22.7%, respectively (Table 1). Residues 46–59 of RanBP3 bound to the major NLS-binding site in an extended conformation were identified unambiguously in the electron density map (

Acknowledgments

We thank Hidemi Hirano for technical assistance and discussion. We thank the staff of Photon Factory for assistance during X-ray diffraction data collection.

Conflict of interest

The authors have no conflict of interest to declare.

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