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• W2000542458 abstract "Article15 October 2003free access Structural basis for Nup2p function in cargo release and karyopherin recycling in nuclear import Yoshiyuki Matsuura Yoshiyuki Matsuura MRC Laboratory of Molecular Biology, Hills Road, Cambridge, CB2 2QH UK Search for more papers by this author Allison Lange Allison Lange Department of Biochemistry, Emory University School of Medicine, Atlanta, GA, 30322-3050 USA Search for more papers by this author Michelle T. Harreman Michelle T. Harreman Department of Biochemistry, Emory University School of Medicine, Atlanta, GA, 30322-3050 USA Search for more papers by this author Anita H. Corbett Anita H. Corbett Department of Biochemistry, Emory University School of Medicine, Atlanta, GA, 30322-3050 USA Search for more papers by this author Murray Stewart Corresponding Author Murray Stewart MRC Laboratory of Molecular Biology, Hills Road, Cambridge, CB2 2QH UK Search for more papers by this author Yoshiyuki Matsuura Yoshiyuki Matsuura MRC Laboratory of Molecular Biology, Hills Road, Cambridge, CB2 2QH UK Search for more papers by this author Allison Lange Allison Lange Department of Biochemistry, Emory University School of Medicine, Atlanta, GA, 30322-3050 USA Search for more papers by this author Michelle T. Harreman Michelle T. Harreman Department of Biochemistry, Emory University School of Medicine, Atlanta, GA, 30322-3050 USA Search for more papers by this author Anita H. Corbett Anita H. Corbett Department of Biochemistry, Emory University School of Medicine, Atlanta, GA, 30322-3050 USA Search for more papers by this author Murray Stewart Corresponding Author Murray Stewart MRC Laboratory of Molecular Biology, Hills Road, Cambridge, CB2 2QH UK Search for more papers by this author Author Information Yoshiyuki Matsuura1, Allison Lange2, Michelle T. Harreman2, Anita H. Corbett2 and Murray Stewart 1 1MRC Laboratory of Molecular Biology, Hills Road, Cambridge, CB2 2QH UK 2Department of Biochemistry, Emory University School of Medicine, Atlanta, GA, 30322-3050 USA *Corresponding author. E-mail: [email protected] The EMBO Journal (2003)22:5358-5369https://doi.org/10.1093/emboj/cdg538 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The yeast nucleoporin Nup2p is associated primarily with the nuclear basket of nuclear pore complexes and is required for efficient importin-α:β-mediated nuclear protein import as well as efficient nuclear export of Kap60p/importin-α. Residues 1–51 of Nup2p bind tightly to Kap60p and are required for Nup2p function in vivo. We have determined the 2.6 Å resolution crystal structure of a complex between this region of Nup2p and the armadillo repeat domain of Kap60p. Nup2p binds along the inner concave groove of Kap60p, but its interaction interface is different from that employed for nuclear localization signal (NLS) recognition although there is some overlap between them. Nup2p binds Kap60p more strongly than NLSs and accelerates release of NLSs from Kap60p. Nup2p itself is released from Kap60p by Cse1p:RanGTP only in the presence of the importin-β binding (IBB) domain of Kap60p. These data indicate that Nup2p increases the overall rate of nuclear trafficking by coordinating nuclear import termination and importin recycling as a concerted process. Introduction Eukaryotic cells transport proteins and RNAs into and out of the nucleus through nuclear pore complexes (NPCs), which are constructed from a large number of different proteins collectively termed nucleoporins (Nups) (Weis, 2003). NPCs have a central disc with 8-fold rotational symmetry embedded in the nuclear envelope together with extensions that form cytoplasmic filaments and nuclear baskets. In Saccharomyces cerevisiae, NPCs are constructed from ∼30 different Nups with a combined mass of 50 MDa (Rout et al., 2000). Many Nups are symmetrically distributed about the central axis of the NPC, although a subset is localized exclusively to the cytoplasmic or nuclear faces and so may be crucial for regulating initiation and termination of trafficking. Nuclear transport is a signal-mediated active process that depends on concerted interactions between nucleoporins, soluble transport factors and their cargo macromolecules. Transport factors (such as karyopherins) recognize cargo in one compartment, translocate it through NPCs, and then release it in the other compartment before being recycled to participate in further rounds of transport (reviewed by Weis, 2003). The classical nuclear localization signal (NLS) is characterized by one or two short stretches of basic residues (Dingwall and Laskey, 1998). In the cytoplasm, NLS-containing proteins bind the Kap60p/importin-α adapter, which, in turn, binds Kap95p/importin-β via its importin-β binding (IBB) domain. This cargo:carrier complex is then docked to and translocated through NPCs in a process mediated by interactions between Kap95p/importin-β and Nups that contain phenylalanine–glycine (FG) sequence repeats (Rexach and Blobel, 1995; Bayliss et al., 2000). Once in the nucleus, RanGTP dissociates the cargo:carrier complex and the importins are recycled to the cytoplasm. Kap95p/importin-β returns bound to RanGTP, whereas recycling of Kap60p/importin-α is mediated by CAS/Cse1p complexed with RanGTP (Kutay et al., 1997; Hood and Silver, 1998; Solsbacher et al., 1998). In the cytoplasm, the Ran GTPase is activated by RanGAP, dissociating the importin-β:RanGTP and importin-α:CAS:RanGTP complexes, freeing the importins for another cycle. Finally, the RanGDP generated in the cytoplasm is recycled by NTF2 to the nucleus where it is recharged with GTP by RCC1. Crystal structures of several key players in the classical NLS-import cycle have been obtained (Conti and Izaurralde, 2001). Kap95p/importin-β is built from 19 α-helical HEAT repeats, whereas Kap60p/importin-α has an N-terminal importin-β binding (IBB) domain, a central armadillo (Arm) repeat domain, and a short C-terminal extension. Arm repeats are constructed from three α-helices and pack into a right-handed superhelix to produce a gently curved elongated molecule (Conti et al., 1998). Both HEAT and Arm repeats provide a structural platform that mediates protein–protein interactions along its surface. The classical NLS sits on the concave inner surface of Kap60p/importin-α, in a groove lined with conserved Trp and Asn residues. There are two NLS binding sites: monopartite NLSs bind primarily to the major site formed by Arm repeats 2–4, whereas bipartite NLSs bind to both this site and a second one, formed by Arm repeats 7–8 (Conti et al., 1998; Conti and Kuriyan, 2000; Fontes et al., 2000). Translocation through NPCs appears to be a reversible facilitated diffusive process and the disassembly/assembly of cargo:carrier complexes in the appropriate compartments not only imparts directionality, but also allows accumulation of cargo against a concentration gradient (Gorlich et al., 2003; Weis, 2003). Import cargoes dissociate from the carriers in the nucleus after which the carriers are recycled to the cytoplasm. The precise way in which cargo is released in the nucleus has not been established unequivocally. The spontaneous dissociation of a classical NLS from the Kap60p:Kap95p heterodimer is quite slow (Gilchrist et al., 2002). This probably prevents premature release of cargo during the transit across the NPC, but necessitates a mechanism by which cargo release is accelerated in the nucleus (Gilchrist et al., 2002). Possible acceleration mechanisms include competition with the auto-inhibitory region of the IBB domain of Kap60p/importin-α (Kobe, 1999; Catimel et al., 2001; Harreman et al., 2003a), competition with the CAS:RanGTP complex (Kutay et al., 1997), and the action of peripheral Nups such as Nup2p (Solsbacher et al., 2000; Gilchrist et al., 2002). Nup2p has a multi-domain structure based on an N-terminal Kap60p binding and NPC targeting domain, a central domain containing 16 tandem FxFG sequence repeats that bind Kap95p, and a C-terminal Ran-binding domain (Loeb et al., 1993). Nup2p has little regular secondary structure (Denning et al., 2002). It is localized primarily to the nucleoplasmic face of NPCs (Solsbacher et al., 2000; Dilworth et al., 2001) and is proposed to form part of the nuclear basket, although in some conditions it can also shuttle between nucleus and cytoplasm (Dilworth et al., 2001). The precise mechanism underlying Nup2p function has not been established, although Gilchrist et al. (2002) have shown that it accelerates the release of the Cbp80p NLS from Kap60p:Kap95p. Lindsay et al. (2002) have suggested that Nup2p may be analogous to vertebrate Npap60, which is proposed to bind the importin:substrate complex and accompany it during import through NPCs. Although Nup2p is not essential in normal genetic backgrounds, yeast lacking Nup2p exhibit defects in Kap60p/Kap95p-mediated nuclear protein import and defects in Cse1p-mediated recycling of Kap60p (Booth et al., 1999; Hood et al., 2000; Solsbacher et al., 2000). Because of its multi-domain structure, Nup2p could mediate a variety of protein–protein interactions, raising the possibility that disassembly and assembly of transport complexes, which are often considered in isolation, could occur in a cooperative fashion in vivo. Here we show that residues 1–50 of Nup2p are required for function in vivo and use X-ray crystallography to define the interaction interface between Nup2p and Kap60p. Nup2p and NLSs have different yet partially overlapping binding sites on Kap60p and the affinity of Nup2p for Kap60p is higher than that of NLSs. Moreover, we present evidence that Nup2p residues 1–51 increase the off-rate of NLS-cargoes from Kap60p and that Nup2p is itself removed from Kap60p by the concerted action of Cse1p:RanGTP and the IBB domain. The unique structural features of the Nup2p:Kap60p interaction, together with these binding and kinetic data, provide insight into the cooperative mechanism by which Nup2p together with the IBB domain, Cse1p and RanGTP orchestrates the efficient release of NLS-cargo in the nucleus concomitant with assembly of the export complexes (Kap95p:RanGTP and Kap60p:Cse1p:RanGTP) required for recycling. Results The N-terminus of Nup2p is required for function in vivo Hood et al. (2000) showed deletion of residues 1–175 inhibited Nup2p targeting to the nuclear envelope as well as binding to Kap60p and suggested a role for the Nup2p N-terminus in providing an initial NPC docking site along the Cse1p:Ran-mediated Kap60p export pathway. Subsequent work suggested that the first 50 residues of Nup2p were critical for Kap60p binding (Denning et al., 2001). To assess the functional importance of these residues, we created a plasmid in which Δ50nup2p (Nup2p residues 51–720) was fused at its C-terminus to GFP. Deletion of residues 1–50 (Δ50nup2p) did not affect the expression of Nup2p (data not shown). The truncated protein was still targeted to the nuclear rim although there was some increased signal in the cytoplasm (Figure 1A). To test the functional importance of Nup2p residues 1–50 in vivo, the Δ50nup2p plasmid, a control wild-type Nup2p plasmid, or vector alone were transformed into Δnup2 srp1-31 double mutant cells (where Nup2p is absolutely essential for viability; Booth et al., 1999) and the SRP1 maintenance plasmid removed by plasmid shuffle (Figure 1B). Although expression of full-length Nup2p complemented Δnup2 srp1-31 cells, expression of Δ50nup2p did not, consistent with residues 1–50 of Nup2p being functionally necessary in vivo. Expression of a construct that expressed only residues 1–51 (1–51nup2p) did not complement the Δnup2 srp1-31 mutant (Figure 1B) and, although a fusion of these residues to GFP was localized to the nucleus, it did not localize specifically to the nuclear rim (data not shown). These data demonstrate that residues 1–50 of Nup2p are necessary, but not sufficient for Nup2p function in vivo. Figure 1.In vivo functional analysis of NUP2. (A) Nup2p and Δ50Nup2p expressed as C-terminal GFP fusions under the control of NUP2 promoter in Δnup2 yeast cells show nuclear envelope localization as visualized by GFP fluorescence. Corresponding DIC and DAPI images are shown. (B) Δnup2 srp1-31 yeast cells maintained by a plasmid encoding Kap60p and expressing either full-length Nup2p, 1–51 Nup2p or Δ50Nup2p were spotted onto control plates lacking uracil or 5-FOA plates. 5-FOA eliminates the URA3 maintenance plasmid encoding Kap60p. (C) The Nup2p N-terminus promotes docking of Kap60p to the nuclear envelope and is required for efficient recycling of Kap60p to the cytoplasm. Kap60p–GFP was integrated at the endogenous SRP1 locus of Δnup2 yeast cells. The cells were then transformed with plasmids encoding either full-length Nup2p, Δ50Nup2p, or vector alone, and Kap60p–GFP was visualized by GFP fluorescence. Corresponding DIC and DAPI images are shown. Download figure Download PowerPoint We exploited the observation that Kap60p accumulates within the nucleus of cells that lack Nup2p (Booth et al., 1999; Hood et al., 2000; Solsbacher et al., 2000) to probe whether residues 1–50 of Nup2p are required for Nup2p-dependent recycling of Kap60p to the cytoplasm. In wild-type cells, integrated Kap60p–GFP was localized to the nuclear rim whereas, in Δnup2 cells, it accumulated within the nucleus. The Δnup2 cells were subsequently transformed with plasmids encoding full-length Nup2p, Δ50nup2p, or vector alone, co-stained with DAPI to mark the position of the nucleus, and visualized (Figure 1C). Integrated Kap60p–GFP was concentrated at the nuclear rim in cells expressing Nup2p, as described (Booth et al., 1999; Solsbacher et al., 2000), but was localized throughout the nucleus of Δnup2 cells expressing Δ50nup2p (or vector alone), consistent with residues 1–50 of Nup2p being essential for efficient recycling of Kap60p to the cytoplasm. Binding assays indicated that Δ50nup2p does not bind Kap60p (Denning et al., 2001) and that residues 1–51 of Nup2p retained the ability to bind tightly to Kap60p (Table I and see below), indicating that the effects seen in vivo were due, at least in part, to decreased interaction between Nup2p and Kap60p. Table 1. Dissociation constants determined by microtitre plate binding assay Kap60p construct Binding partner KD (nM) 1–542, wild-type GST-Nup2p (1–720), wild-type 4.4 ± 1.1 88–530, wild-type GST-Nup2p (1–720), wild-type 0.07 ± 0.02 88–530, wild-type GST-Nup2p (1–720), R38A/R39A 3.0 ± 0.4 88–530, wild-type GST-Nup2p (1–720), R47A/R48A 0.13 ± 0.02 88–530, wild-type GST-Nup2p (1–720), R38A/R39A/R47A/R48A 240 ± 60 1–542, wild-type GST-Nup2p (1–51), wild-type 30 ± 5 88–530, wild-type GST-Nup2p (1–51), wild-type 2.1 ± 0.3 88–530, Y397D GST-Nup2p (1–51), wild-type 4.4 ± 0.5 88–542, wild-type GST-Nup2p (1–51), wild-type 2.4 ± 0.4 88–542, wild-type GST-Nup2p (1–174), wild-type 3.5 ± 0.4 88–542, wild-type GST-Nup2p (36–51), wild-type 20 ± 1 BFP-81–542, wild-type GST-Nup2p (1–51), wild-type 2.2 ± 0.4 1–542, wild-type GST-SV40 NLS 1000 ± 400 88–542, wild-type GST-SV40 NLS 22 ± 3 BFP-81–542, wild-type SV40 NLS-GFP 12 ± 2 1–542, wild-type GST-nucleoplasmin NLS 450 ± 100 88–542, wild-type GST-nucleoplasmin NLS 34 ± 4 BFP-81–542, wild-type nucleoplasmin NLS-GFP 9 ± 1 1–542, wild-type GST not detectable 88–542, wild-type GST not detectable 88–530, wild-type GST not detectable 88–530, Y397D GST not detectable BFP-81–542, wild-type GST not detectable BFP-81–542, wild-type GFP not detectable Data represent the best-fit value ± standard error as analyzed by nonlinear regression assuming one site binding. Each assay was performed in duplicate. Crystal structure of Nup2p residues 1–51 complexed with Kap60Δ We used X-ray crystallography to address the structural basis of the interaction between Kap60p and Nup2p. The IBB domain of Kap60p is not required for Nup2p binding (Booth et al., 1999; Hood et al., 2000). A truncated construct of Kap60p (residues 88–530; Kap60Δ) bound tightly to full-length Nup2p (KD 0.07 ± 0.02 nM) and a Nup2p N-terminal fragment (residues 1–51; Nup2N) retained nM affinity (KD 2.1 ± 0.3 nM) for Kap60Δ (Table I). The Kap60Δ mutant Y397D that has improved solubility (Conti and Kuriyan, 2000) had comparable affinity for Nup2N (KD 4.4 ± 0.5 nM) and was used for crystallization. We obtained P21212 crystals of the Kap60Δ:Nup2N complex that diffracted to 2.6 Å resolution and solved their structure by molecular replacement. The final model was refined to a free R-factor of 25.7% (R-factor 21.6%) and contained residues 88–526 of Kap60p, residues 36–51 of Nup2p and 158 waters (Table II). The two Kap60Δ:Nup2N complexes in the asymmetric unit were essentially identical, indicating that crystal packing interactions had not significantly altered their conformation. Although the two Kap60Δ chains in the asymmetric unit formed a symmetrical dimer with roughly one third of the N-terminal portion of each contributing to the dimer interface, each Nup2N chain interacted almost exclusively with a single Kap60Δ. Gel filtration indicated that the Kap60Δ:Nup2N complex existed primarily as a monomer in solution (data not shown), indicating that the dimer in the crystals was probably not important physiologically. The Kap60Δ chains in the complex were similar to those in the Kap60Δ:SV40-NLS or Kap60Δ:nucleoplasmin-NLS complexes (Conti et al., 1998; Conti and Kuriyan, 2000): the overall Cα r.m.s. deviations were 0.67 Å and 1.23 Å, respectively, indicating that the binding of Nup2N was not accompanied by a major conformational change. Table 2. Crystallographic statistics Data collection statistics Space group P21212 Unit cell dimensions (Å) a = 129.81, b = 140.08, c = 63.99 Resolution range (Å)a 20–2.6 (2.74–2.60) Mosaicity 0.75 Total observationsa 76810 (10875) Unique reflectionsa 29670 (4308) Completeness (%)a 97.5 (98.3) Rmerge (%)a 8.6 (53.1) I/σa 10.0 (1.8) Refinement statistics Number of reflections (working, test) 33802/1806 Rcryst/Rfree (%) 21.6/25.7 Total number of non-H atoms 7189 Number of water molecules 158 R.m.s. deviation from ideal bond length (Å) 0.012 R.m.s. deviation from ideal bond angles (degree) 1.831 Ramachandran plot (%) Core region 92.6 Allowed region 7.0 Generously allowed region 0.4 Disallowed region 0.0 aParentheses refer to final resolution shell. The interface between Kap60Δ and the Nup2p N-terminus The interaction interface between Nup2p and Kap60Δ was different to that seen with NLSs or the IBB domain (Kobe, 1999; Conti and Kuriyan, 2000). The extensive region of Nup2p visible in the omit map (Figure 2A) contained an elongated region (residues 36–45) that ran along the inner groove of Kap60Δ, together with a type I β-turn (residues 46–50), which reversed the direction of the chain towards the Kap60Δ C-terminus. Nup2p packed against the third helices of Arm repeats 4–8 and was intimately attached at two distinct sites. The first was located between the major and minor NLS binding sites and involved primarily interactions between residues 43–51 of Nup2p and Arm repeats 4–6, whereas the second site corresponded to the minor NLS binding site and involved primarily interactions between Nup2p residues 36–40 and surface pockets in Arm repeats 7 and 8. The Nup2p chains were well defined, with average B-factors of 47 and 56 Å2 reducing to generally 30–50 Å2 at the binding sites. The buried interfacial area was 2393 Å2 and involved a combination of H-bonds, electrostatic interactions and stereospecific hydrophobic interactions. The path of the Nup2p main chain on Kap60Δ partially overlapped with that of the N-terminal half of the bipartite nucleoplasmin NLS, but its overlap with the monopartite c-myc or SV40 NLS was less severe (Figure 2B). Figure 2.Structure of residues 1–51 of Nup2p bound to Kap60Δ. (A) Overview of the Kap60Δ:Nup2N complex. The Fo − Fc omit map corresponding to the Nup2p fragment contoured at 2.5 σ and the refined model of Nup2p (residues 36–51) are superimposed. (B) Overlay of Nup2p with NLS peptides. The coordinates of Kap60Δ bound to Nup2N (blue) and the SV40 (purple), c-myc (yellow), and nucleoplasmin (green) NLSs (Conti et al., 1998; Conti and Kuriyan, 2000) were superimposed, then removed to show the relative positions of each ligand. Orientation as in (A). (A and B) were prepared with Bobscript (Esnouf, 1997) and Raster3D (Merritt and Bacon, 1997). (C) Molecular surface of Kap60Δ coloured by electrostatic potential shaded from −13 kT/e (red) to +13 kT/e (blue) (calculated with Nup2N removed using GRASP; Nicholls et al., 1991) shows acidic pockets and a nonpolar surface that recognize Nup2p. Nup2p residues are bold. Download figure Download PowerPoint The Nup2p residues contributing to the first binding site included an IBB-like sequence, 45KRR47, together with flanking hydrophobic residues. However, the Nup2p KRR sequence bound in a novel way (Figures 2C and 3) that was quite different from that observed for the IBB domain (Kobe, 1999). Thus, the side chain of Nup2p Met43 was inserted into a hydrophobic pocket around Kap60Δ Tyr283 and the interaction was further stabilized by a H-bond between the OH of Tyr283 and the Nup2p backbone. The side chain of Kap60Δ Arg321 formed H-bonds with the main chain carbonyls of Nup2p Ala42 and Met43, holding Met43 in an appropriate position and orientation for insertion of its hydrophobic side chain into the nonpolar pocket. The side chain of Nup2p Arg47 extended into the P1 acidic pocket in the major NLS binding site of Kap60Δ (Conti et al., 1998; Conti and Kuriyan, 2000) where its guanidinium group formed H-bonds with Ser240, Asn241 and Asp276. There is an electrostatically neutral region on the surface of Kap60Δ between the major and minor NLS binding sites (Figure 2C), and this hydrophobic patch (centred on Trp279) made interactions with the aliphatic regions of the side chains of Nup2p Lys45, Arg46 and Arg47 as well as with Phe50. The side chains of Nup2p Met43, Arg47 and Phe50 were well defined in the electron density map and so were probably key determinants of binding specificity and affinity. Figure 3.Schematic comparison of the interactions of Kap60Δ with (A) Nup2p, (B) IBB domain (based on mouse importin α; Kobe, 1999), (C) monopartite NLS (Conti et al., 1998), and (D) bipartite NLS (Conti and Kuriyan, 2000). Download figure Download PowerPoint At the second Nup2p binding site, Nup2p Met36 and Arg38 fit into the two pockets in the minor NLS binding site in much the same way as observed for a bipartite NLS (Conti and Kuriyan, 2000). The side chain of Arg38 that occupied the P2′ pocket had well defined density and formed a H-bond with Kap60Δ Glu402 as well as a cation-π interaction with the ring of Trp405. The side chains of Kap60Δ Asn367 and Trp363 formed H-bonds with the main chain amide and carbonyl of Nup2p Arg38, stabilizing the interactions at this second site. The side chains of Nup2p Arg39 and Lys40 did not have strong electron density, but did point towards acidic potentials on the Kap60Δ surface around Asp286 and Glu360 (Figure 2C), and so probably make additional contributions to binding. To evaluate the contribution of different Nup2p residues to Kap60p binding, we constructed full-length Nup2p mutants in which either of the two Arg clusters (Arg38/39 and Arg46/47) or all four Args were mutated to Ala, and measured binding to Kap60Δ (Table I). Full-length Nup2p bound (KD 0.07 ± 0.02 nM) ∼30-fold more strongly than Nup2N, indicating that residues C-terminal to Lys51 also contribute to the interaction. This additional contribution probably derived from the FxFG repeats and/or the Ran binding domain, because residues 1–174 had an affinity for Kap60Δ (KD 3.5 ± 0.4 nM) comparable to Nup2N (KD 2.4 ± 0.4 nM). In this context it is interesting to note that in the Kap60Δ:Nup2N structure, many hydrophobic Nup2p side chains (Ile41, Ala42, Pro44, Met48 and Ala49) are exposed to solvent and so part of the rest of Nup2p may pack over them, reducing the entropic penalty associated with binding Kap60Δ. R38/39A mutations in Nup2p weakened Kap60Δ binding by ∼40-fold (KD 3.0 ± 0.4 nM) whereas the R46/47A mutations were less effective (KD 0.13 ± 0.02 nM). When combined, however, the mutations were synergistic and the affinity of the R38/39/46/47A mutant for Kap60Δ was reduced by three orders of magnitude (KD 240 ± 60 nM). These data show that both Arg clusters are important for the interaction and support the idea that the N-terminal 51 residues of Nup2p represent the primary Kap60p binding site. Although Nup2p residues 1–35 did not have clear electron density, they probably made an additional, minor, contribution to binding because residues 36–51 alone had 10-fold weaker affinity (KD 20 ± 1 nM) for Kap60Δ. The interaction between Kap60Δ and Nup2p is different from the interaction with either classical NLSs or the IBB domain In Kap60:NLS structures, clusters of basic residues bind in the major NLS binding site in an extended conformation (Conti et al., 1998; Conti and Kuriyan, 2000; see Figures 2B and 3). This contrasts with the Kap60Δ: Nup2N interaction, where the downstream KRR sequence in Nup2p interacts in a novel way, leaving most of the major NLS binding site accessible (Figure 3). Site-directed mutagenesis confirmed the differences between the binding sites of Nup2p and NLSs on Kap60Δ. Thus, whereas the D203K/N157A mutation in the primary NLS binding site of Kap60Δ abolished binding to the SV40 NLS, Nup2N binding was retained (Figure 4A). Three factors probably contributed to these differences. First, Nup2p residues 36–38 are specifically anchored at the minor NLS site; secondly, the linker sequence between the upstream and downstream basic clusters of Nup2p is shorter than in bipartite NLSs; and thirdly, the hydrophobic residues flanking the KRR sequence allow Nup2p residues 46–50 to form a β-hairpin and pack against a nonpolar patch on Kap60Δ. Figure 4.Nup2p–Kap60p interactions are different from NLS interactions and Nup2p competes with NLSs. (A) N157A/D203K mutations in the major NLS binding site of Kap60Δ abolish SV40 NLS binding but not Nup2N binding. GST–Nup2N (10 μg; lanes 1 and 3) or GST–SV40 NLS (10 μg; lanes 2 and 4) was treated with 15 μg of Kap60Δ. (B) Nup2N competes both monopartite and bipartite NLSs from Kap60Δ. Beads containing 4.5 μg GST–SV40 NLS or GST–nucleoplasmin (NP) NLS were treated with Kap60Δ (10 μg) which remained mainly bound after washing with binding buffer but was subsequently removed by 30 μM His/S–Nup2N. (C) NLS-cargoes do not effectively compete Nup2N from Kap60Δ and only bind weakly in the presence of Nup2N. GST––Nup2N (4.5 μg) was treated with Kap60Δ (10 μg) ± 30 μM NLS–GFP (lanes 1–3) or 30 μM NLS–GFP alone (lanes 4 and 5). Download figure Download PowerPoint Nup2N accelerates dissociation of NLSs from Kap60Δ As illustrated in Figure 3, although the Nup2p binding site on Kap60p is different from both NLS binding sites, there is a degree of overlap. Nup2p would clash with monopartite NLSs in the P1 pocket of the major NLS binding site and so could destabilize interaction with neighbouring pockets, particularly the P2 site crucial for NLS recognition (Hodel et al., 2001). Although the bipartite nucleoplasmin NLS does not use the P1 site, it would clash with Nup2p at the minor NLS site and also at the intervening region. Furthermore, Nup2N bound more strongly to Kap60Δ (KD ∼2.4 nM) than the SV40 or nucleoplasmin (NP) NLS (KD ∼10–30 nM; Table I). Consequently, the Nup2p residues 1–51 should compete NLSs from Kap60p. This prediction was confirmed by an equilibrium cross-competition assay (Figure 4B and C), consistent with a previous study using Nup2p residues 1–174 (Solsbacher et al., 2000). Equilibrium competition could result from either active displacement or passive competition, which can be discriminated by examining the influence of Nup2p on the rate of dissociation of NLSs from Kap60p. Gilchrist et al. (2002) have shown that full-length Nup2p increases the off-rate of Cbp80p-NLS from Kap95p:Kap60p:NLS and Kap60p:NLS, supporting an active displacement mechanism. We used a fluorescence resonance energy transfer (FRET)-based assay to show that Nup2p residues 1–51 are sufficient to accelerate the release of both monopartite (SV40) and bipartite (NP) NLSs from Kap60Δ without the need for Kap95p or RanGTP. Blue fluorescent protein (BFP, donor) was fused to the N-terminus of Kap60Δ and GFP (acceptor) was fused to the C-terminus of each NLS. As expected from the close proximity of the NLS C-terminus and the Kap60Δ N-terminus in the complex (Conti and Kuryan, 2000), a powerful BFP–GFP FRET signal at 510 nm was produced when NLS–GFPs were added to BFP–Kap60Δ (Figure 5A, line 3). This signal decreased on addition of Nup2N (Figure 5A, line 4), consistent with its replacing the NLSs. No FRET decrease was seen when 1 mg/ml BSA was added and no FRET signal was seen when GFP alone was added to BFP-Kap60Δ (data not shown). BFP–Kap60Δ had normal affinity for Nup2N and NLS-GFP fusions (Table I), confirming that the fusions did not interfere with these interactions. Figure 5B shows the dissociation kinetics of the BFP–Kap60Δ:SV40-NLS–GFP complex. The spontaneous dissociation rate (koff of 0.07/s) was estimated from the decrease in the complex's FRET signal in a 20-fold molar excess of Kap60Δ (Figure 5B) to prevent rebinding and was not increased at higher Kap60Δ concentrations (data not shown). Adding Nup2N to the complex dramatica" @default.
• W2000542458 created "2016-06-24" @default.
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• W2000542458 date "2003-10-15" @default.
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• W2000542458 title "Structural basis for Nup2p function in cargo release and karyopherin recycling in nuclear import" @default.
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