Biochemical and Biophysical Research Communications
Crystal structure of importin-α3 bound to the nuclear localization signal of Ran-binding protein 3
Graphical abstract
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.
References (33)
The Ran GTPase: theme and variations
Curr. Biol.
(2002)- et al.
Human RanBP3, a group of nuclear RanGTP binding proteins
FEBS Lett.
(1998) - et al.
Ran-binding protein 3 links Crm1 to the Ran guanine nucleotide exchange factor
J. Biol. Chem.
(2002) - et al.
Structural insights into how Yrb2p accelerates the assembly of the Xpo1p nuclear export complex
Cell Rep.
(2014) - et al.
Nuclear export of Smad2 and Smad3 by RanBP3 facilitates termination of TGF-beta signaling
Dev. Cell
(2009) - et al.
Biophysical characterization of interactions involving importin-alpha during nuclear import
J. Biol. Chem.
(2001) - et al.
Ran-binding protein 3 phosphorylation links the Ras and PI3-kinase pathways to nucleocytoplasmic transport
Mol. Cell
(2008) - et al.
AKT activation drives the nuclear localization of CSE1L and a pro-oncogenic transcriptional activation in ovarian cancer cells
Exp. Cell Res.
(2013) - et al.
Molecular determinants for nuclear import of influenza A PB2 by importin alpha isoforms 3 and 7
Structure
(2015) - et al.
Structural biology and regulation of protein import into the nucleus
J. Mol. Biol.
(2016)
Molecular determinants for nuclear import of influenza A PB2 by importin alpha isoforms 3 and 7
Structure
RanBP3 influences interactions between CRM1 and its nuclear protein export substrates
EMBO Rep.
Ran-binding protein 3 is a cofactor for Crm1-mediated nuclear protein export
J. Cell Biol.
RanBP3 enhances nuclear export of active (beta)-catenin independently of CRM1
J. Cell Biol.
RanBP3 contains an unusual nuclear localization signal that is imported preferentially by importin-alpha3
Mol. Cell Biol.
Transport between the cell nucleus and the cytoplasm
Annu. Rev. Cell Dev. Biol.
Cited by (9)
Nuclear-Import Receptors Counter Deleterious Phase Transitions in Neurodegenerative Disease: Nuclear-import receptors combat deleterious phases
2022, Journal of Molecular BiologyCitation Excerpt :Ideally, compounds could be uncovered that restore the affinity of Kapβ2 for disease-linked cargo to similar levels observed with wild-type cargo. Although structures of Impα/Kapβ1 bound to the TDP-43 NLS are not yet available, other structures of Impα family members bound to cargo have been solved,118–123 and these could also inform drug design. Here, it will be important to ensure that small-molecules do not make NIR-cargo interactions so tight that they cannot be dissociated by Ran-GTP, as release of cargo is essential for restoring nuclear activity and NIR recycling.16
Deciphering the Binding of the Nuclear Localization Sequence of Myc Protein to the Nuclear Carrier Importin α3
2022, International Journal of Molecular Sciences