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Originally published In Press as doi:10.1074/jbc.M709093200 on January 9, 2008

J. Biol. Chem., Vol. 283, Issue 12, 7877-7884, March 21, 2008
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Molecular Basis for the Recognition of Snurportin 1 by Importin β*Formula

Gregory Mitrousis, Adam S. Olia, Nancy Walker-Kopp, and Gino Cingolani1

From the Department of Biochemistry and Molecular Biology, State University of New York (SUNY) Upstate Medical University, Syracuse, New York 13210

Received for publication, November 6, 2007 , and in revised form, December 24, 2007.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The nuclear import of uridine-rich ribonucleoproteins is mediated by the transport adaptor snurportin 1 (SNP1). Similar to importin {alpha}, SNP1 uses an N-terminal importin β binding (sIBB) domain to recruit the receptor importin β and gain access to the nucleus. In this study, we demonstrate that the sIBB domain has a bipartite nature, which contains two distinct binding determinants for importin β. The first determinant spans residues 25-65 and includes the previously identified importin {alpha} IBB ({alpha}IBB) region of homology. The second binding determinant encompasses residues 1-24 and resembles region 1011-1035 of the nucleoporin 153 (Nup153). The two binding determinants synergize within the sIBB domain to confer a low nanomolar binding affinity for importin β (Kd ~ 2 nM) in an interaction that, in vitro, is displaced by RanGTP. We propose that in vivo the synergy of Nup153 and nuclear RanGTP promotes translocation of uridine-rich ribonucleoproteins into the nucleus.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Nuclear import of proteins and nucleic acids is an active signal-mediated process that requires, in most cases, soluble transport factors and GTP hydrolysis by the GTPase Ran (1-4). Nuclear transport factors are sorted into two categories: importins and exportins (also known as karyopherins), which include 14 members in budding yeast and at least 20 in humans (5, 6). All karyopherins fold into superhelical solenoids, which present an external convex surface involved in nucleoporin binding and a concave internal face that interacts with transport cargos and RanGTP. The two best characterized importins, human importin β1 and yeast karyopherin β2, have been visualized using crystallographic methods bound to specific import cargos (7-10) and RanGTP (11, 12). These structures have provided invaluable information on the structural flexibility of karyopherins and their ability to undergo conformational changes during the import process (13-16).

The best understood nuclear import pathway involves cargos bearing a classical SV40-like nuclear localization sequence (PKKKRKV) (17). These cargos are imported into the nucleus by the adaptor importin {alpha} and the receptor importin β with the aid of RanGTP. GTP hydrolysis by Ran energizes the import reaction by promoting both release of the import complex from nucleoporins as well as unloading of import cargos into the nucleus (1-3). In addition to classical nuclear localization sequence cargos, uridine-rich ribonucleoproteins (U snRNPs)2 represent an important class of import cargos. Mature U snRNP particles are assembled in the cytoplasm and imported into the nucleus in at least two distinct pathways, both dependent on importin β (18). In the first pathway, the import signal is in the proteinaceous core of the U snRNP formed by the Sm proteins (18-21). In the second pathway, the trimethylated guanosine cap of the mature U snRNP is recognized by the adaptor snurportin 1 (SNP1) (18-21), which, in turn, recruits importin β. SNP1 consists of an N-terminal IBB domain (sIBB) similar to that found in importin {alpha} ({alpha}IBB) and a large C-terminal trimethylated guanosine cap-binding region that resembles the GTP-binding domain of mRNA-guanylyltransferase (22). In permeabilized cells, SNP1 and importin β promote Ran- and energy-independent nuclear import of at least two specific spliceosomal U snRNPs, namely U1 and U5 (23). After nuclear entry, SNP1 is recycled back to the cytoplasm by Crm1 in complex with RanGTP (24). In this study, we have used a combination of crystallographic, biochemical, and biophysical techniques to define the composition and recognition of the sIBB domain by importin β.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Biochemical Techniques—Human importin β1 (residues 1-876) was expressed and purified as described previously in Ref. 8. HEAT repeats 1-10 of importin β (residues 1-445) were cloned into the NdeI and NotI sites of the pTYB2 vector (New England Biolabs, Inc.) and expressed in the Escherichia coli ER2566 strain. Purification of importin β-(1-445) was performed as described for the full-length protein in Ref. 25. The IBB constructs sIBB-(1-65), sIBB-(25-65), and {alpha}IBB-(11-54) were amplified by PCR from an SNP1 (19) or hSRP1{alpha} (26) template and ligated into a unique NcoI site of the pGEX-4T vector. The construct sn-(1-24) was generated by introducing a stop codon at position 25 of the plasmid pGEX-sIBB-(1-65). GST-sIBBs and GST-{alpha}IBB constructs were expressed in E. coli BL21 (DE3) strain for 3 h at 30 °C after induction with 0.5 mM isopropyl-1-thio-β-D-galactopyranoside and purified over GST beads followed by gel filtration chromatography. Ran was expressed, purified, and loaded with the non-hydrolyzable GTP analog, GppNHp, as described previously (27).

Crystallization and Structure Determination—Importin β bound to a chemically synthesized sIBB-(25-65) peptide was crystallized under 20% polyethylene glycol 8000, 50 mM sodium chloride, at pH 6.0. Crystals of the importin β-sIBB-(25-65) complex diffracted weakly to 5Å resolution. Prolonged dehydration of crystals by soaking in 38% polyethylene glycol 8000 dramatically improved the diffraction to 2.35 Å resolution. Approximately 50 crystals were soaked for various times and screened at beamline X6A at the Brookhaven National Light Source (BNLS) on a Quantum Q4 CCD detector. Diffraction data were reduced to intensities using the programs DENZO and SCALEPACK (28) (see Table 1). Two distinct crystal forms were obtained, both in space group P21. Crystal form I (see ds2.35 in Table 1) contains one importin β-sIBB-(25-65) complex in the asymmetric unit. Crystal form II (see ds3.2 in Table 1), which diffracted weakly to 3.2 Å resolution, contains two importin β-sIBB-(25-65) complexes in different conformations per asymmetric unit. Both structures were solved by molecular replacement in MolRep (29) using human importin β (Protein Data Bank (PDB) code 1QGK) as a search model. The initial solution was refined in CNS (30) using rigid body refinement, simulated annealing, and grouped B-factor refinement to an Rfree ~ 35% (calculated using 10% of the observed reflections). The sIBB-(25-65) domain was built in Fo - Fc electron density difference maps using the program Coot (31). After several rounds of manual building alternated with positional and B-factor refinement, the final model for crystal form I has a Rwork and Rfree of 22.7 and 25.0%, respectively. For crystal form II, the final model was refined to a Rwork of 30.3% and Rfree of 32.8%, using all reflections between 40 and 3.2 Å resolution and includes a well defined closed importin β-sIBB domain complex (complex A) and an open conformation of the complex (complex B). All structural figures were made using PyMOL (32).


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  		TABLE 1
X-ray data collection and refinement statistics for importin β -sIBB-(25-65) complexes

The numbers in parentheses refer to the statistics for the outer resolution shell: (2.43-2.35 Å) for data sets ds2.35 and (3.31-3.20 Å) for data set ds3.2.

 
Native Gel Electrophoresis and Pull-down Assay—Native binding assay on agarose gel was carried out as described in Ref. 33. In the assay, 25 µg of importin β, a gel filtration-purified complex of importin β bound to either sIBB-(1-65) or sIBB-(25-65) peptides, were separated on a 1.5% agarose gel at room temperature for 45 min. For the Ran displacement, gel filtration-purified importin β-sIBB-(1-65) or sIBB-(25-65) complexes were incubated with 0.25-5-fold molar excess of RanGppNHp and incubated for 30 min on ice prior to electrophoretic separation on agarose gel. The pull-down assay was performed on glutathione-agarose beads (Amersham Biosciences) coupled to GST-sIBB-(1-65). In the assay, 25 µl of GST-sIBB-(1-65) beads were incubated with 16 µg of importin β for 15 min at room temperature. For Ran displacement, an excess of either RanGppNHp or RanGDP was added to the beads after the addition of importin β. The mixtures were incubated overnight at 4 °C followed by extensive washing in pull-down buffer (20 mM HEPES, pH 7.4, 150 mM sodium chloride, 3 mM β-mercaptoethanol, 0.005% Tween 20). After washing, all samples were dissolved in SDS-loading buffer and analyzed on SDS-PAGE.

Isothermal Titration Calorimetry (ITC)—ITC experiments were carried out at 30 °C in a ITC calorimeter (Microcal). sn-(1-24) dissolved in β-buffer (10 mM HEPES, pH 7.4, 150 mM sodium chloride, 3 mM EDTA, 3 mM β-mercaptoethanol) at a concentration of 450 µM was injected in 9-µl increments into the calorimetric cell containing 1.8 ml of importin β in β-buffer at a concentration of 60 µM. The spacing between injections was 360 s. Titration data were analyzed using the Origin 7.0 data analysis software (Microcal Software, Northampton, MA). Injections were integrated following manual adjustment of the baselines. Heats of dilution were determined from control experiments with the β-buffer and subtracted prior to curve fitting using a single set of binding sites model. The curve fitting yields a Kd =~30 ± 14 µM and {Delta}H = 6948 ± 1491 cal/mol at 30 °C for titration of the sn-(1-24) peptide into full-length importin β and a Kd =~30 ± 15 µM and {Delta}H = 6548 ± 1191 cal/mol for titration of the sn-(1-24) peptide into importin β (residues 1-445).

Surface Plasmon Resonance (SPR)—GST control, GST-sIBB-(25-65), GST-sIBB-(1-65), and GST-{alpha}IBB-(11-54) were captured onto a sensor chip using an immobilized anti-GST antibody. Importin β was flowed into the cell in β-buffer (plus 0.005% surfactant P20) at concentrations between 200 and 600 nM, at a flow rate of 60 µl/min. Data were analyzed using the BIAevaluation software (Biacore Life Sciences). The association and dissociation curves were fit separately using the simple 1:1 Langmuirian model. The analysis of each individual curve resulted in an apparent rate of association and dissociation kon = 7.22 e4 ± 1.8 e4 (1/ms), koff = 8.24 e-4 ± 0.5 e-4 (s-1), and kon = 1.01 e5 ± 0.3 e4 (1/ms), koff = 2.07 e-4 ± 0.3 e-4 (s-1) for sIBB-(25-65) and sIBB-(1-65), respectively. The dissociation constants of the sIBB-(25-65) and sIBB-(1-65) for importin β measured as Kd = koff/kon was Kd = 15.6 ± 5.8 nM and Kd = 2.0 ± 0.4 nM, respectively.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Snurportin IBB Domain Is Bipartite—Nuclear import SNP1 is mediated by the N-terminal sIBB domain (residues 1-65) (19). This region alone is necessary and sufficient to promote nuclear import in the absence of Ran and energy in a permeabilized cell assay (23). Analysis of the primary sequence of the sIBB domain suggests the presence of a bipartite signal (Fig. 1a). Region 25-65 of the sIBB domain closely resembles the classical IBB domain of importin {alpha} ({alpha}IBB), which promotes Ran/energy-dependent nuclear import (23, 34). Similar to the {alpha}IBB domain (residues 11-54), all basic residues critical for importin β binding are conserved in the sIBB-(25-65) domain (supplemental Fig. 1). The latter is, however, shorter due to a 3-amino-acid gap between residues 46 and 47 (Fig. 1a). The region 25-65 of the sIBB domain will be defined as the {alpha}IBB region of homology. In contrast to importin {alpha}s, which show poor sequence conservation upstream of the IBB domain, SNP1 residues 1-24, sn-(1-24), are well conserved in snurportins (supplemental Fig. 1). Blast analysis reveals that sn-(1-24) shares 42% sequence identity and 79% similarity to a region of Nup153 spanning residues 1011-1035. This same region of Nup153 binds importin β with high affinity, likely via interactions with phenylalanine-glycine (FXFG) repeats (25).


Figure 1
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  		FIGURE 1.
Structure of the sIBB domain. a, the sIBB domain spans residues 1-65 of SNP1 and contains two structural determinants: region 1-24, sn-(1-24), has 42% identity to region 1011-1035 of Nup153, whereas region 25-65 is 55% identical to the IBB domain of importin {alpha}1 (hSRP1{alpha}) (26). In the alignment, identical residues are colored in red. b, left panel, ribbon diagram of crystal form I of importin β (in green) bound to the sIBB-(25-65) domain (in magenta), determined at 2.35 Å resolution. Right panel, blowup of the sIBB-(25-65) domain.

 
Structure of Importin β Bound to the sIBB-(25-65) Domain—To shed light on the interaction of SNP1 with importin β, we crystallized human importin β bound to a chemically synthesized peptide spanning residues 25-65 of SNP1. Although crystals of the importin β-sIBB-(25-65) complex were obtained under the same conditions used for crystallization with the {alpha}IBB-(11-54) peptide (8), crystals diffracted weakly to 5 Å resolution. Aiming at high resolution diffraction studies, we subjected the importin β-sIBB-(25-65) complex crystals to dehydration in highly concentrated solutions of polyethylene glycol 8000. Diffraction analysis confirmed a dramatic improvement in the diffraction quality and resolution limit as a function of the time of dehydration. Dehydration for 3-12 h yielded high quality diffraction past 2.3 Å resolution. This crystal form, referred to as crystal form I, displayed a small primitive monoclinic unit cell (Table 1), which contained one importin β-sIBB-(25-65) complex in the asymmetric unit and ~55% solvent content. The structure of crystal form I was determined by the molecular replacement method and refined to an Rfree of 25.0%, including all reflections between 40 and 2.35 Å resolution (Fig. 1b and Table 1). In the structure, importin β adopts a closed conformation nearly identical to that seen in complex with the {alpha}IBB domain (8). The protein is built by 19 HEAT repeats, which wrap around the sIBB-(25-65) in a conformation reminiscent of a snail. The sIBB-(25-65) domain interacts with the concave surface of importin β between HEAT repeats 7-19. The sIBB-(25-65) domain folds as a long C-terminal helix (residues 41-65), connected to an N-terminal 310 helix (residues 27-30) by a 7-residue spoon-shaped linker (Fig. 1b). The overall B-factor for the peptide is ~65 Å2, slightly higher than that for the importin β (~45 Å2).


Figure 2
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  		FIGURE 2.
Structural plasticity of importin β bound to the sIBB-(25-65) domain. a, ribbon diagram of the asymmetric unit content of crystal form II, determined at 3.2 Å resolution. The two complexes in the asymmetric unit have different conformations. In complex B, on the left (in cyan), importin β adopts an open conformation, and only the sIBB-(40-65) helix is visible (in yellow). In complex A, on the right (in green), importin β has a conformation identical to crystal form I, and all residues for the sIBB-(25-65) domain are visible (in magenta). b, superimposition of the importin β structures from complex A (closed) and B (open) reveals deviations up to 20 Å in the C terminus of the protein.

 
Crystal form II was obtained upon short dehydration (~30 min) of importin β-sIBB-(25-65) crystals and diffracted to ~3.2 Å resolution. Interestingly, this second crystal form contains two importin β-sIBB-(25-65) complexes in different conformations per asymmetric unit (Fig. 2a). In one complex (referred to as complex A), importin β adopts a closed conformation nearly identical to crystal form I. In the second complex (complex B), importin β is distinctly open, with C-terminal HEAT repeats 12-19 swung up to 20 Å away from the corresponding position in the closed conformation (Fig. 2b). The tertiary structure of the importin β molecule in complex B resembles Kap95p, the yeast homologue of importin β, in complex with RanGTP (11). This open conformation of importin β likely represents the structure adopted by the protein in the process to unload the import cargo. Accordingly, the electron density for the sIBB-(25-65) domain is less defined in the complex B, where only the C-terminal helix is visible.

sIBB-(25-65) versus {alpha}IBB Domain—The structure of importin β bound to the {alpha}IBB-(11-54) domain was previously determined to a comparable resolution of 2.3 Å (8), allowing for direct comparison with the crystal form I described in this study. Despite the profound functional differences between the sIBB and {alpha}IBB domain, the conformation adopted by importin β in the two complexes is virtually identical (r.m.s. deviation 0.965 Å) (Fig. 3a). It is noteworthy that significant differences exist in the structure of the two IBB domains and in the way importin β positions them inside the cargo-binding surface (Fig. 3a). First, the sIBB-(25-65) domain is slightly shorter than the {alpha}IBB domain (35 versus 41 Å), which is consistent with the 3-amino-acid gap in the C-terminal helix between residues 46 and 47 (Fig. 1a). Second, the sIBB C-terminal helix is ~5° tilted with respect to the {alpha}IBB helical axis, which positions it closer to the importin β concave surface (Fig. 3a). Third, in the sIBB-(25-65) domain, the linker between the C-terminal helix and the N-terminal 310 helix is significantly longer than in the {alpha}IBB domain (7 versus 3 amino acids), which suggests in the sIBB domain that these two helices have a much higher degree of flexibility. This hypothesis is corroborated by the structural plasticity seen in the 3.2 Å crystal form II, where in the more open complex B (Fig. 2a) only the C-terminal helix of the sIBB domain is visible, whereas the N-terminal 310 helix is likely flexible and thus disordered in the crystal structure.

As in the recognition of the {alpha}IBB-(11-54) domain, importin β makes two distinct sets of contacts with the sIBB domain. The first includes the N-terminal moiety of the sIBB peptide (residues 25-40), which interacts with HEAT repeats 7-11 (Fig. 3b). This region is critical for binding of SNP1 to importin β. Substitution of Arg27 to Ala decreases the affinity of SNP1 for importin β by 20-fold and blocks nuclear import in digitonin-permeabilized cells (35). The second region of interaction involves the sIBB helix (residues 41-65), which presents an extended network of contacts with HEAT repeats 12-19 of importin β (Fig. 3b). In the sIBB domain, this region engages in fewer contacts, 12 versus 20, as compared with the {alpha}IBB helix (residues 26-54). Likewise, the polybasic stretch 27Arg-Arg-Arg-Arg31 of the {alpha}IBB domain is shifted in the sIBB helix where only 2 of the 4 arginines contact importin β directly (Fig. 3b).

SNP1 Region (1-24) Contains a Binding Determinant for Importin β—We next sought to determine whether the Nup153-like moiety of SNP1, sn-(1-24), binds importin β directly. The interaction of the sn-(1-24) with importin β was probed using three independent binding techniques. First, by native electrophoresis on agarose gel, a chemically synthesized sn-(1-24) peptide was found to shift the migration of importin β, which is suggestive of a direct physical interaction between the peptide and the protein (Fig. 4a). Second, using ITC, titration of sn-(1-24) in a calorimetric cell containing importin β at 30 °C yielded an endothermic binding reaction that is adequately described by a single exponential decay binding model (Fig. 4b). The thermodynamic parameters obtained from the curve fit indicated that sn-(1-24) binds importin β with a Kd ~ 30 ± 14 µM and an enthalpy of complex formation of {Delta}H = 6948 ± 1491 cal/mol. Interestingly, identical heat release (and thus Kd) was observed using a fragment of importin β comprising HEAT repeats 1-10 (residues 1-445) (Fig. 4c). These evidences lend support to the existence of a single binding site for the sn-(1-24) in the N-terminal, RanGTP-(11, 27), and nucleoporin-binding (36) region of importin β. However, based on the ITC data, we cannot rule out the possibility that the sn-(1-24) binds with lower or similar affinity to the C terminus of importin β (HEAT 11-19) or that the binding of the sn-(1-24) to the N-terminal HEAT repeats 1-10 excludes or drastically decrease its binding to the C terminus of the protein. Third, using SPR, we found that the sIBB-(1-65) domain has a 7-fold higher binding affinity for importin β than the shorter sIBB-(25-65) domain lacking the sn-(1-24) (Kd ~ 2.0 ± 0.5 nM versus 15.2 ± 5.8 nM) (Fig. 5). The affinity measured by SPR for the full-length sIBB-(1-65) domain agrees well with the Kd values measured by FRET (Kd ~ 2.0 nM) (37) and solid phase binding assay (Kd ~ 4.7 nM) (35), which makes us confident in the accuracy of this technique in measuring the interactions of importin β with the SNP1. In addition to a significant drop in the Kd, the sIBB-(25-65) domain had a 4-fold faster rate of dissociation from importin β as compared with the sIBB-(1-65) domain (koff = 8.24 e4 ± 0.5 e4 s-1 versus koff = 2.07 e4 ± 0.3 e4 s-1) (Fig. 5), whose off rate from importin β was comparable with that of importin {alpha} (38).


Figure 3
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  		FIGURE 3.
sIBB-(25-65) domain versus {alpha}IBB-(11-54) domain. a, left panel, structure of importin β-{alpha}IBB complex (in purple and blue, respectively) superimposed to that of the importin β-sIBB-(25-65) complex (in green and magenta, respectively) of crystal form I. Right panel, blowup of the {alpha}IBB and sIBB-(25-65) domains (colored in blue and magenta, respectively) translated out of the superimposition. Significant differences are observed in the structure of the two peptides. b, schematic diagram of the interactions between HEAT repeats 7-19 of importin β and the sIBB-(25-65) domain (top) as compared with the {alpha}IBB-(11-54) domain (bottom). HEAT repeats are referred to as H7-19. Colored in red in the primary sequence of the sIBB-(25-65) and {alpha}IBB-(11-54) domain are identical residues. Intermolecular polar and hydrophobic interactions are shown as red and green lines, respectively. Intramolecular contacts within IBB domains are indicated by red brackets.

 
RanGppNHp Displaces the sIBB Domain from Importin β—We next investigated whether the interaction between SNP1 and importin β is disrupted by the small GTPase Ran, in vitro. By pull-down assay, agarose beads coupled to GST-sIBB-(1-65) efficiently pulled-down importin β (Fig. 6, lane 4). This interaction was selectively disrupted by an excess of RanGpp-NHp as compared with RanGDP (Fig. 6, lanes 5 and 6, respectively). In a second set of experiments, we sought to determine whether the N-terminal moiety sn-(1-24) alters the affinity of Ran for the importin β-sIBB complex. To address this question, we isolated on a Superdex 200 gel filtration column a 1:1 complex of importin β bound to either the sIBB-(1-65) or the sIBB-(25-65) domain. The stoichiometric presence of sIBB peptides was confirmed on Tricine gel (data not shown). Increasing quantities of Ran preloaded with non-hydrolyzable GTP analog, RanGppNHp, were titrated into the preformed gel filtration-purified importin β-sIBB-(25-65) or importin β-sIBB-(1-65) complex, and the mixture was analyzed by native electrophoresis on agarose gel (Fig. 7). In agreement with the pull-down assay, RanGpp-NHp selectively dissociated both sIBB-(25-65) and sIBB-(1-65) domains from importin β as compared with RanGDP (supplemental Fig. 2). Although both peptides were dissociated by approximately equimolar quantities of RanGpp-NHp, the dissociation of the shorter sIBB-(25-65) domain lacking the sn-(1-24) appeared reproducibly less cooperative than the sIBB-(1-65) domain (in Fig. 7, a versus b, compare lanes 4-8). This may suggest that importin β becomes more sensitive to RanGppNHp binding when the SPN1 moiety sn-(1-24) is bound to its N-terminal domain.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
To determine the molecular basis for the sIBB-(1-65) karyopheric properties, we have dissected the structure of the sIBB-(1-65) domain and its binding interactions with importin β. The main conclusion of our work indicates that the sIBB-(1-65) domain has a bipartite organization, which consists of two moieties, both interacting with importin β. The first binding determinant of SNP1 spans residues 25-65 and includes an {alpha}IBB region of homology, which binds importin β with nanomolar affinity (Kd ~ 15 nM). As shown in the crystal structure of the importin β bound to the sIBB-(25-65) domain, this region of SNP1 interacts with HEAT repeats 7-19 of importin β similar to an {alpha}IBB domain and not only with the N terminus of the protein (residues 1-618), as reported previously (20). Within the sIBB-(25-65) domain, the C-terminal helix (residues 41-65) makes fewer and weaker contacts with importin β than the N-terminal residues 25-40 of SNP1. This observation partially explains why an N-terminal fragment of importin β (residues 1-618) shows relatively high binding affinity for the sIBB domain (20, 35), but it fails to bind the {alpha}IBB domain (39). Likewise, the paucity of contacts between the sIBB helix (residues 41-65) and importin β may explain the fast rate of dissociation measured by SPR between importin β and the sIBB-(25-65) domain as compared with its counterpart, the {alpha}IBB domain (38).


Figure 4
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  		FIGURE 4.
sn-(1-24) contains a binding determinant for importin β. a, native electrophoresis on agarose gel. Lane 1, free importin β; lane 2, importin β in the presence of a 3-fold excess of sn-(1-24) peptide. b and c, ITC analysis of the interaction of the sn-(1-24) peptide with importin β. ITC titration of the sn-(1-24) peptide into a calorimetric cell containing either full-length importin β (residues 1-876) (b) or a C-terminally truncated fragment of importin β spanning residues 1-445 (c) was performed. In both b and c, the raw data are in the top panel, and the integrated enthalpy plotted as a function of the sn-(1-24):importin β molar ratio is shown in the bottom panel. The dissociation constants measured from the ITC data for the interactions sn-(1-24)-importin β and sn-(1-24)-importin β-(1-445) are Kd = 30 ± 14 µM and Kd = 30 ± 15 µM, respectively.

 
In addition to the {alpha}IBB region of homology, SNP1 presents a novel binding determinant for importin β between residues 1 and 24. This region interacts weakly with importin β (Kd ~ 30 µM) when isolated from SNP1 but synergizes with the sIBB-(25-65) moiety in the context of the sIBB-(1-65) domain to confer low nanomolar binding affinity and a slow rate of dissociation from importin β. Therefore, we propose that the sn-(1-24) acts as a molecular hook to retain SNP1 bound to importin β. Interestingly, sn-(1-24) shares high sequence identity to the region 1011-1035 of Nup153, which is localized at the nuclear basket (40) and is known to bind importin β with nanomolar affinity (25). This prompts the intriguing idea of a direct displacement of the sn-(1-24) from importin β within the snRNP-SNP1-importin β import complex at the nuclear basket. Notably, this scenario is conceptually analogous to what was reported for the yeast nucleoporin Nup2p, which is also associated with the nuclear basket (41, 42). Residues 1-51 of Nup2p bind tightly to Kap60p, the yeast homologue of importin {alpha}, and thus accelerate the release of nuclear localization sequence cargos from Kap60p. In addition, the region 1-24 of SNP1 was also shown to be critical for binding to Crm1, which, in complex with RanGTP, recycles SNP1 back to the cytoplasm (24).

How do our data fit with the observation that in digitonin-permeabilized cells, the sIBB domain promotes Ran- and energy-independent nuclear import (23)? The structural and biochemical work presented in this study challenges the idea of a simple Ran-independent nuclear import pathway in at least three ways. First, at the three-dimensional level, the conformation of importin β bound to the sIBB domain is very similar to that adopted in complex with the {alpha}IBB domain, which requires RanGTP to undergo a dramatic conformational change necessary to release the import cargo (11). Second, the sIBB domain binds importin β with low nanomolar binding affinity, which is incompatible with a Ran-independent cargo release into the nucleus. Third, in vitro, RanGTP, and not RanGDP, specifically disrupts the importin β-sIBB complex. To complement these data, we provide evidences that region 1-24 of SNP1 contains a Nup153-like motif that modulates both the strength of interaction and the off rate of the sIBB domain from importin β. We propose that the sn-(1-24) plays an important functional role during the import reaction by reducing the avidity of the NPC for the sIBB-import complex. This could be explained, for instance, by a simple intermolecular contact between the SNP1 moiety sn-(1-24), which contain a 12FSVS15 repeat, and the major nucleoporin-binding site in importin β (25). Such an interaction would reduce the avidity of the sIBB-import complex for nucleoporins and possibly allow translocation of the import complex through the NPC in permeabilized cells, in the absence of exogenous RanGTP. The idea of a reduced avidity of the NPC for the sIBB-import complex agrees well with the observation that a chimera of the sIBB-(1-65) fused to the β-galactosidase, sIBB-β-galactosidase, remains associated for less time to the nuclear basket than an {alpha}IBB-β-galactosidase, suggesting a reduced stalling of the sIBB-import complex inside the NPC (20).


Figure 5
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  		FIGURE 5.
sn-(1-24) affects the off rate of the sIBB domain from importin β. Binding properties of sIBB-(25-65) domain and sIBB-(1-65) domain were compared using SPR. GST-sIBB-(25-65), GST-sIBB-(1-65), and GST-{alpha}IBB-(11-54) were captured onto a sensor chip using an immobilized anti-GST antibody. Importin β was flown into the cell between concentrations of 200-600 nM, at a flow rate of 60 µl/min. The SPR curves shown are from three identical experiments carried out using importin β at 600 nM.

 


Figure 6
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  		FIGURE 6.
RanGppNHp and not RanGDP disrupts the importin β-sIBB-(1-65) complex. Importin β (lane 1) is efficiently pulled down by glutathione agarose beads coupled to GST-sIBB-(1-65) (lanes 3 and 4, respectively). Incubation of this complex with an excess of RanGppNHp selectively displaces importin β from GST-sIBB-(1-65) (lane 5) with respect to a control where beads where incubated with RanGDP (lane 6).

 


Figure 7
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  		FIGURE 7.
RanGppNHp releases the sIBB domain from importin β independent of the sn-(1-24). a and b, electrophoretic mobility shift assay on native agarose gel. Free importin β and importin β bound to RanGppNHp are in lanes 1 and 2, respectively. Gel filtration-purified complexes of importin β bound to sIBB-(25-65) domain (panel a) or sIBB-(1-65) domain (panel b) are in lane 3. The addition of increasing quantities of RanGppNHp (from 0.25- to 5-fold molar excess) to either importin β-sIBB complex displaces the sIBB domains from importin β (lanes 4-8).

 
However, if the reduced avidity of the sIBB-import complex for the NPC may be sufficient to explain why in permeabilized cells the sIBB domain translocates efficiently without the addition of exogenous Ran, it does not explain in an in vivo setting what energizes the active release (and thus nuclear import) of sIBB-import cargos into the cell nucleus. In agreement with the observation that in vitro the sIBB-importin β interaction is specifically disrupted by RanGTP, we propose that GTP hydrolysis by the small GTPase Ran remains the driving force of the dissociation of SNP1 from importin β. Further studies are needed to better understand this important import pathway. In particular, it will be necessary to develop a functional assay that allows discrimination between nuclear passage of the sIBB-import complex and dissociation from importin β, and that defines the energetic requirement of these two processes.


   FOOTNOTES
 
The atomic coordinates and structure factors (code 2P8Q, 2Q5D) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

* This work was supported by National Institutes of Health Grant GM074846. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

Formula The on-line version of this article (available at http://www.jbc.org) contains two supplemental figures. Back

1 To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, SUNY Upstate Medical University, 750, E. Adams St., Syracuse, NY, 13210. Tel.: 315-464-8744; Fax: 315-464-8750; E-mail: cingolag{at}upstate.edu.

2 The abbreviations used are: U snRNP, uridine-rich small nuclear ribonucleoprotein; SNP1, snurportin 1; IBB, importin β binding; sIBB, SNP1 IBB; {alpha}IBB, importin {alpha}; NPC, nuclear pore complex; Nup153, nucleoporin 153; ITC, isothermal titration calorimetry; SPR, surface plasmon resonance; GST, glutathione S-transferase. Back


   ACKNOWLEDGMENTS
 
We thank Wanda Coombs for assistance with the SPR measurements. We are grateful to Vivian Stojanoff and the staff at National Synchrotron Light Source beamline X6A as well as to the macCHESS staff for assistance in data collection.



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 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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