Structural Basis for the Recognition of a Nucleoporin FG Repeat by the NTF2-like Domain of the TAP/p15 mrna Nuclear Export Factor

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1 Molecular Cell, Vol. 8, , September, 2001, Copyright 2001 by Cell Press Structural Basis for the Recognition of a Nucleoporin FG Repeat by the NTF2-like Domain of the TAP/p15 mrna Nuclear Export Factor Sébastien Fribourg, Isabelle C. Braun, Elisa Izaurralde, and Elena Conti 1 European Molecular Biology Laboratory Meyerhofstrasse 1 D Heidelberg Germany Summary TAP-p15 heterodimers have been implicated in the export of mrnas through nuclear pore complexes (NPCs). We report a structural analysis of the interac- tion domains of TAP and p15 in a ternary complex with a Phe-Gly (FG) repeat of an NPC component. The TAPp15 heterodimer is structurally similar to the homodi- meric transport factor NTF2, but unlike NTF2, it is incompatible with either homodimerization or Ran bind- ing. The NTF2-like heterodimer functions as a single structural unit in recognizing an FG repeat at a hy- drophobic pocket present only on TAP and not on p15. This FG binding site interacts synergistically with a second site at the C terminus of TAP to mediate mrna transport through the pore. In general, our findings suggest that FG repeats bind with a similar conformation to different classes of transport factors. Introduction (trnas or U snrnas, for example) in the presence of RanGTP in the nucleus, dock to the NPC, and release it in the cytosol upon GTP hydrolysis. Nuclear import of Ran proceeds instead via a small homodimeric protein known as NTF2 or p10, which is not related to the importin- -like family (Ribbeck et al., 1998). Studies of mrna export are converging on a family of transport factors known as the nuclear export factor (NXF) family, which is also unrelated to the karyopherin family. The yeast genome encodes a single NXF protein known as Mex67p, and multiple NXF homologs are present in higher eukaryotes (Segref et al., 1997; Herold et al., 2000). S. cerevisiae Mex67p and the C. elegans NXF1 protein have been shown to be essential for the nuclear export of bulk polyadenylated RNAs (Segref et al., 1997; Tan et al., 2000). The human NXF1 homolog TAP was originally identified as the cellular factor recruited by simian type D retroviruses to export their unspliced genomic RNA, which contains the so-called constitutive transport element (CTE) to which TAP binds (Grüter et al., 1998; Braun et al., 1999; Kang and Cullen, 1999). More recently, TAP has been found to directly stimulate the nuclear export of cellular mrnas that are otherwise exported inefficiently (Braun et al., 2001). TAP and its S. cerevisiae and C. elegans homologs have also been shown to interact with FG-containing nucleoporins (Ka- tahira et al., 1999; Bachi et al., 2000; Strä er et al., 2000; Tan et al., 2000), thus exhibiting two of the essential properties of an mrna export factor: cargo binding and NPC binding. To export mrna cargoes, TAP must associate with p15 (a small protein also known as NXT1; Braun et al., 2001), which shares significant sequence homology with the RanGDP nuclear import factor NTF2 (Black et al., 1999; Katahira et al., 1999; Suyama et al., 2000). NXF proteins are conserved in eukaryotes. Their mrna export function is conserved to such an extent that overexpression of human TAP-p15 heterodimers partially restores growth of the otherwise lethal mex67/ mtr2 double knockout strain in yeast (Katahira et al., 1999). Overexpression of TAP alone is unable to rescue the similarly lethal mex67 knockout phenotype, sug- gesting that human p15 and yeast Mtr2p might be functional analogs recognized by NXF proteins in a speciesdependent manner. The p15 and Mtr2p proteins share no obvious sequence similarity but interact with homolo- gous regions in human TAP and yeast Mex67p (Herold et al., 2000; Strä er et al., 2000; Suyama et al., 2000). NXF proteins have a modular domain organization. In human TAP, the N-terminal half (residues 1 372) con- Nucleocytoplasmic transport factors shuttle efficiently through the channels spanning the nuclear envelope (nuclear pore complexes; NPCs), carrying their protein or RNA cargoes specifically at a high rate (Mattaj and Englmeier, 1998; Pemberton et al., 1998; Nakielny and Dreyfuss, 1999; Görlich and Kutay, 1999). Transport factors have translocation-promoting properties re- sulting from their ability to interact with the proteins lining the NPC, collectively known as nucleoporins. In particular, the phenylalanine-glycine (FG) repeats that characterize most nucleoporins have been shown to interact with several transport factors in yeast twohybrid assays, in vitro and in vivo (Ryan and Wente, 2000). Although the actual molecular mechanism of transport through the NPC is controversial, the nucleop- orin FG repeats are believed to function as docking sites for the transport factors moving through the pore (Rout et al., 2000; Ben-Efraim and Gerace, 2001; Ribbeck and Görlich, 2001). The bulk of nucleocytoplasmic trafficking is sustained by the family of karyopherin/importin- -like proteins. tains a nuclear localization signal (Bear et al., 1999; The importin- -like transport factors have a common Bachi et al., 2000), an RNA binding (RNP) domain, and structural framework and respond to the small GTPase a leucine-rich repeat (LRR) domain whose structure we Ran to bind or release their cargo within the appropriate have already characterized (Liker et al., 2000). The RNP cellular compartments (Weis, 1998; Conti and Izaurralde, domain is required to bind and export the viral CTE RNA, 2001). Importins bind their protein cargo in the cytosol, but is dispensable for cellular mrna export and is the and after NPC translocation, release it in the nucleus least conserved domain in the NXF family (Braun et al., upon binding RanGTP. Exportins bind their cargo 1999; Liker et al., 2000). The LRR domain is conserved and essential for TAP-mediated export of both viral CTEcontaining RNAs and cellular mrnas (Liker et al., 1 Correspondence: conti@embl-heidelberg.de 2000;

2 Molecular Cell 646 Braun et al., 2001). While the CTE RNA interacts directly rectly by forming a single structural and functional unit with a fragment of TAP containing the RNP and LRR with the NTF2-like domain of TAP. Our results indicate domains, several lines of evidence suggest that the in- that the NPC binding domain of TAP (residues ) teraction of TAP with cellular mrna cargoes is probably has two FG nucleoporin binding sites, one in the NTF2- not direct, but mediated by additional proteins (Braun like domain and one in the UBA-like domain. We show et al., 1999; Bachi et al., 2000; Liker et al., 2000; Strä er that each FG binding site contributes to NPC associaand Hurt, 2000; Stutz et al., 2000). tion, and that both are required and act synergistically The C-terminal half of TAP (residues ) consists to export cellular mrnas. Mutation of both FG binding of two domains, one of which has low but detectable sites destroys nuclear rim association and mrna export sequence similarity to NTF2 and is required for the inter- activity, resulting in a dominant-negative effect. action with p15 (Katahira et al., 1999; Bachi et al., 2000; Suyama et al., 2000), and the other which is predicted Results and Discussion to have a ubiquitin-associated (UBA) fold (Suyama et al., 2000). In human TAP and C. elegans NXF1, mutations Structural Overview or deletion of the UBA-like domain ( UBA) strongly re- The crystal structure of the C-terminal half of human TAP duce both NPC association in vivo and nucleoporin complexed to p15 was determined using MAD (multiple binding in vitro (Bachi et al., 2000; Herold et al., 2000; anomalous dispersion) phasing on a mercury derivative. Suyama et al., 2000; Tan et al., 2000). As a result, the Although the crystallized fragment of TAP included resimrna export activity of the TAP UBA mutant is mark- dues , the C-terminal portion (residues ) edly reduced, but not abolished (Braun et al., 2001). In forming the putative UBA-like domain was disordered. S. cerevisiae, the analogous Mex67p UBA mutant has With the exception of a small loop (residues ), a temperature-sensitive phenotype and accumulates the p15-interacting domain of TAP could be completely poly(a) RNA in the nucleus (Strä er et al., 2000). Dele- traced in the electron density, as could the entire p15 tion of the UBA-like domain in S. cerevisiae Mex67p protein (residues 2 140). The final model of the complex prevents its localization at the nuclear rim, but not its has been refined to 1.9 Å; resolution with an R free of ability to bind FG repeat nucleoporins in vitro. Mutagen- 22.5% (Table 1). esis studies suggest that FG binding to Mex67p is medi- The structures of p15 and the p15-interacting domain ated by the NTF2-like domain and is dependent on Mtr2p of TAP have a highly curved sheet flanked by helices, (Strä er et al., 2000). On the other hand, yeast Mex67p forming a conical molecule (Figure 1A). As predicted can also interact directly with FG nucleoporins in the (Black et al., 1999; Katahira et al., 1999; Suyama et al., absence of Mtr2p (Strawn et al., 2001), as similarly can 2000), p15 is structurally similar to NTF2. The two prowild-type human TAP in the absence of p15 (Bachi et teins have 21% sequence identity and 113 pairs of topoal., 2000). Given these apparently discordant results, logically equivalent carbon atoms, which can be suthe molecular mechanism of NPC association of NXF perposed with an rms deviation of 1.9 Å (Figure 2). The homologs and in particular the function of the essential TAP domain is also structurally homologous to NTF2, NTF2-like domains of TAP and p15 is unclear. albeit to a lesser extent. The similarity involves 95 C In this study, we address the questions of whether atoms, which superpose with an rms deviation of 2.5 Å, the NTF2-like domains predicted in human TAP and p15 but only 13% of the structurally aligned residues are contribute to NPC translocation of mrna cargoes and chemically identical (Figure 2). whether they have similar NPC-associating properties The most striking difference between the matching to the NTF2 transport factor. Mapping of the nucleoporin domains of NTF2, p15, and TAP is the presence of a binding properties of the RanGDP import factor NTF2 by large and unexpected insertion loop of 33 residues after site-directed mutagenesis has identified a hydrophobic the first strand of TAP (Figures 1A and 2). This insertion surface patch that affects NPC association, and thereloop contains two short helices, 2A and 2B, whose fore RanGDP import efficiency (Bayliss et al., 1999; Ribconformation is fixed by the interaction of a set of resibeck and Görlich, 2001), but no structural information is available at present on nucleoporin NTF2 recognition. dues (Tyr435, Arg440, Asn441, Glu501, and Arg511) that The molecular insight revealed by the crystal structure is well conserved in TAP and its homologs (Figure 2). of an FxFG repeat bound to importin (Bayliss et al., The domain boundaries of the structurally defined NTF2-2000) can not readily be extrapolated to NTF2, since the like domain of TAP (residues ) coincide with two nucleocytoplasmic transport factors have com- those mapped for the functional interaction with p15 pletely different structures. (Bachi et al., 2000), but differ from those in the sequence We report the crystal structure determination of a alignment proposed by Suyama et al. (2000) because of complex between p15 and the p15 binding domain of the unexpected insertion loop. TAP at 1.9 Å resolution, and its interaction with a nucleoporin FG-containing peptide at 2.8 Å resolution. Specificity of the NTF2-like TAP-p15 Interaction: TAP and p15 associate to form a heterodimer with a Heterodimerization versus Homodimerization similar overall structure to the NTF2 homodimer. In cona The NTF2-like domains of TAP and p15 interact to form trast to the RanGDP import factor NTF2, the structures compact heterodimer similar to the NTF2-NTF2 homo- of TAP and p15 are incompatible with either Ran binding dimer (Figure 1B). The two NTF2-like domains face each or homodimerization. The heterodimer binds a nucleoptheir other across the convex faces of their sheets, and orin FG repeat sequence in a single hydrophobic pocket secondary structure elements are approximately in the TAP domain. Although p15 itself does not bind related by 2-fold symmetry. The interacting surfaces the FG-containing peptide directly, it contributes indi- are lined not only by hydrophobic but also by hydrophilic

3 FG Nucleoporin Recognition by TAP-p15 Heterodimer 647 Table 1. Data Collection, Phasing and Refinement Statistics TAP-p15 TAP-p15-FG repeat Data collection and phasing statistics Space group P P Cell dimensions (Å) a b c a b c X-ray source ESRF ID14-1 Elettra ESRF ID14-3 (Grenoble, Fr) (Trieste, It) (Grenoble, Fr) Wavelength (Å) Resolution (Å) (remote) 2 (peak) 1 (inflection) Total measurements Unique reflections Redundancy Completeness (%) 99.3 (93.3) 94.4 (78.5) 93.3 (75.7) 92.0 (62.5) 98.1 (99.9) R sym (%) 7.1 (30.0) 4.8 (11.2) 4.0 (7.6) 3.9 (6.8) 5.4 (35.1) FOM (acentric) 0.61 Refinement statistics R free 22.5% 26.9% R working 22.0% 24.0% φ most favored 92.0% 87.9% φ additionally allowed 7.3% 11.8% Rmsd bond Rmsd angle Protein residues Water molecules residues (particularly in the case of p15), which are only we identified an interacting domain in the range of residues weakly conserved and interact indirectly via water molecules in CAN (data not shown). This region The heterodimeric interaction is strengthened by contains five FG repeats. Given that low-affinity interactions tight contacts involving the insertion loop of TAP. with micromolar dissociation constants have been Among the direct TAP-p15 interactions, a salt bridge reported for the binding of FG repeat-containing peptides between Asp482 in TAP and Arg134 in p15 is particularly to other transport factors, including NTF2 and imbetween well conserved (Figure 2). A structurally equivalent salt portin (Bayliss et al., 1999; Chaillan-Huntington et al., bridge is present in the NTF2-NTF2 symmetric homodimer. 2000; Ribbeck and Görlich, 2001), a large (40-fold) ex- This salt bridge is not symmetric in the TAP-p15 cess of a 12 amino acid peptide containing a single FG heterodimer, where Asp76 in p15 interacts instead with repeat was added to the protein for cocrystallization. Arg440 in the TAP insertion loop (Figure 1B). The interac- The structure of the TAP-p15 NTF2-like domain in a tion is strengthened by the invariant TAP Asn441 contacting complex with this FG-containing peptide was deter- p15 main chain atoms and by the invariant p15 mined at 2.8 Å resolution and refined to an R free of 26.9% Gln78 contacting TAP main chain atoms. Amino acids (Table 1). involved in direct contacts between the N-terminal helix The heterodimeric complex of TAP and p15 does not of p15 ( 1) and helix 2B of TAP are not well conserved undergo major conformational changes upon FG repeat and might affect species-dependent recognition. binding, as demonstrated by the very low rms deviation The insertion loop of TAP plays a critical role, not only between the two structures (0.3 Å). Despite the large in the heterodimeric interaction with p15, but also in excess of FG-containing peptide used in the crystallization disfavoring a homodimeric interaction. Helix 2B in particular experiments, the electron density map shows that would clash with the N-terminal strand 0 (Fig- a 1808 Pro-Gly-Phe-Gly-Gln 1812 moiety specifically binds ures 1A and 2) in a putative TAP-TAP homodimer, preventing the NTF2-like heterodimer at a single site near the N its formation. Homodimerization of p15 is also terminus of TAP helix 1 (Figures 1A and 3A). likely to be disfavored because of the long loop connecting The FG-containing peptide folds in a tight turn on the strands 3 and 4. protein surface, with the side chain of the phenylalanine residue protruding into a hydrophobic pocket of TAP. Specific Recognition of a Nucleoporin FG Repeat The aromatic ring stacks against the conserved TAP by the NTF2-like Domain of TAP Pro521 and is surrounded by Leu383, Leu386, Gln486, To investigate the molecular determinants of TAPnucleoporin Leu491, Ala519, and Leu527 (Figures 3A and 3B). The recognition, we carried out cocrystallization downstream glycine of the nucleoporin repeat is in close experiments of the NPC binding portion of the transport contact with TAP Pro521. The side chains of the proline factor with peptides derived from nucleoporin repeats. and glutamine residues flanking the GFG motif are The C-terminal half of TAP, which includes the NTF2- poorly defined in the electron density. Therefore, the like and UBA-like domains, has previously been shown binding of this GFG peptide would be compatible with to interact with several FG repeat-containing nucleoporins, both an FxFG-type or a GLFG-type nucleoporin se- including the nucleoporin CAN, which is found at quence. The structures of TAP-p15 bound to peptides the cytoplasmic fibrils of the NPC (Katahira et al., 1999; containing two contiguous FG repeats (corresponding Bachi et al., 2000). Using GST pull-down experiments, either to residues or residues of

4 Molecular Cell 648 Figure 1. Structures of NTF2-like Domains Involved in Nuclear Transport (A) Ribbon diagram of the NTF2-like domain of TAP (red), p15 (yellow), and NTF2 (green) viewed at their sheet surface. The N and C termini of the NTF2-like domains of TAP correspond to residues 371 and 551. The secondary structure elements are labeled in TAP and are as defined in Figure 2, and the first strand is indicated in p15 and NTF2 for reference. The insertion loop characteristic of TAP is shown in gray, with dots representing a disordered region. Arrows point at the nucleoporin binding site of TAP (present study) and at the RanGDP binding site of NTF2 (Stewart et al., 1998). These and similar pictures were produced using the Ribbons and Setor programs (Carson, 1991; Evans, 1993) (B) Heterodimeric interaction of TAP and p15 (red and yellow, respectively) compared to the homodimeric interaction of NTF2 (green). The structures are shown with the sheet surface edge on, at roughly 90 from the view in (A). An enlargement of the interaction interface in the central panel shows selected side chains of TAP and p15 involved in direct domain-domain interactions. The side chains are in red and yellow for TAP and p15, respectively, while the secondary structure elements are shown in a lighter tint. CAN) are very similar, with only one FG being recognized by the NTF2-like domain (data not shown). No binding of the nucleoporin peptide can be detected at the corresponding position in p15. The surface of p15 is more hydrophilic than that of the TAP NTF2-like domain, and an additional turn of the N-terminal 1 helix occludes the surface to an incoming nucleoporin Phe residue (Figures 1A and 3C). The nuclear import factor NTF2 contains a hydrophobic pocket at a similar location to the FG binding pocket in the TAP NTF2-like domain (Figure 3D). At the position equivalent to TAP Leu383, the NTF2 pocket features the residue Trp7 (Figures 2 and 3D), which has been shown by mutagenesis experiments to be involved in nucleoporin binding (Bayliss et al., 1999). This suggests that an FG binding site is present in each NTF2 monomer at a similar position to the FG binding site of the TAP NTF2- like domain. The recognition of the nucleoporin phenylalanine residue is likely to differ in detail in the two NTF2- like scaffolds, since the arrangements of the residues lining the hydrophobic pockets differ. For example, NTF2 does not have a proline at the position equivalent to TAP residue 521 (Figure 3D). Nucleoporin FG Repeats Bind with a Similar Conformation to Different Transport Factors A comparison of the structures of three different FG repeats bound to TAP and importin (Bayliss et al., 2000; Figures 3B and 3E) shows that the xfg parts of the nucleoporin peptides adopt very similar tight turns (Figure 3F). The turns appear to be facilitated by glycines, one of which in particular has dihedral angles (φ 121, 78 ) that would not be favorable for other amino acids. Apart from the similar main chain conformation, the phenylalanine side chain adopts different rotamer conformations to fit into the hydrophobic pockets of the two transport factors. The phenylalanine binding pockets in the TAP NTF2-like domain and in importin are unrelated and structurally dissimilar. The interaction of the transport factors with what is essentially a single phenylalanine side chain of the nucleoporin repeat raises the question of the specificity

5 FG Nucleoporin Recognition by TAP-p15 Heterodimer 649 Figure 2. Structure-Based Sequence Alignment of NTF2-like Transport Factors Secondary structure elements of human p15 and TAP are shown above and below the corresponding sequences and are represented with cylinders ( helices) and arrows ( strands). The stars indicate residues of p15 and TAP that are structurally equivalent to NTF2 (i.e., their C positions are within 2.5 Å). The structural alignment of TAP (Hs), p15 (Hs), and NTF2 (Rn) was performed using the DALI program (Holm and Sander, 1993). Residues conserved in the homologs from H. sapiens (Hs), C. elegans (Ce), D. melanogaster (Dm), R. norvegicus (Rn), S. cerevisiae (Sc), and S. pombe (Sp) are boxed in yellow. Residues that were mutated in p15 or TAP and analyzed for function are highlighted in red. Previously mutated residues in NTF2 or Mex67p are also indicated in red. The insertion loop of the TAP NTF2-like domain is indicated. of the recognition of Phe-containing nucleoporins as opposed to that of Phe residues on the surface of unrelated proteins. The specificity appears to depend not only on the phenylalanine residue but also on the presence of flanking glycines, which provide sufficient conformational flexibility for the phenylalanine to access the apolar pocket. The generally low affinity of FG repeat nucleoporin binding is not surprising, considering that essentially only one side chain is recognized. Indeed, the high rate of NPC translocation events would be im- possible if the individual interactions were of high affinity (Ribbeck and Görlich, 2001). In vivo, the low affinity is likely to be overcome by the high concentration of FG repeats lining the NPC (Bayliss et al., 1999). The NTF2-like Domain of TAP Is Functional Only in the Presence of p15 The NTF2-like domain of TAP forms a single structural unit with the NTF2-like p15. Using an assay that allows quantification of TAP export activity in cultured cells

6 Molecular Cell 650 Figure 3. FG Nucleoporin Recognition by Transport Factors (A) Recognition of nucleoporin FG repeats by nucleocytoplasmic transport factors. Structure of an FG-containing peptide (in black) bound to the NTF2-like domain of TAP. The 2.8 Å resolution electron density from a simulated annealing omit map was computed after removal of the peptide from the refined model and contoured at 3. The phenylalanine of the peptide inserts its side chain into a pocket formed by a set of residues shown in red. (B) Surface representation showing the hydrophobic FG binding pocket of TAP with the bound nucleoporin repeat peptide. The surface is colored according to the electrostatic potential and viewed in an orientation similar to (A). This figure and similar ones were generated with the program GRASP, with negatively charged areas shown in red and positively charged in blue (Nicholls et al., 1991). (C) The corresponding surface of p15 is more hydrophilic and presents no accessible pocket for FG-containing nucleoporins. (D) NTF2 has a hydrophobic cavity at the equivalent structural position to the FG binding pocket of the NTF2-like domain of TAP in (A). Among the hydrophobic residues that line the pocket (green), Trp7 has previously been shown by mutagenesis experiments to be important for FG nucleoporin binding (Bayliss et al., 1999). (E) Recognition of an FxFG-containing peptide on the importin surface (Bayliss et al., 2000). One phenylalanine residue in particular is inserted into a pocket lined by the residues indicated. (F) Superposition of the x-phe-gly residues of nucleoporin repeats bound to TAP NTF2-like domain (black) and to importin (gray; Bayliss et al., 2000) shows they have a similar conformation when bound to the two different transport factors. The lighter gray corresponds to the FG motif bound at the principal nucleoporin binding site of importin, and corresponds to the structure shown in (E). of TAP Ile518 to Arg destroys the activity almost completely. This mutation is predicted to severely disrupt the heterodimeric interface, since Ile518 is exposed on the sheet surface of TAP and interacts with the sheet surface of p15 (Figures 1B and 4A). An analogous drastic effect is obtained by deleting the entire NTF2-like do- main of TAP (TAP NTF2; Figure 4A), as previously reported (Braun et al., 2001). When the corresponding mutations in p15 (Arg134 to Asp and Ile114 to Arg; Figure 1B) were tested, a reduc- tion of the export activity to only 43% and 30% of the normal value was observed, respectively (Figure 4A). However, these values need to be corrected for the presence of endogenous free p15 in the cultured cells, since overexpression of TAP alone in this assay results (Braun et al., 2001), we investigated the effect of disrupting TAP-p15 heterodimer formation by introducing mutations in both proteins based on the precise localization of residues in the heterodimerization interface. In this assay, human cells are transfected with a reporter plasmid harboring a CAT coding sequence within an inefficiently spliced intron, which is therefore retained within the nucleus. Overexpression of TAP-p15 bypasses nuclear retention and promotes the export of the inefficiently spliced mrna (Braun et al., 2001), leading to about a 40-fold increase of CAT activity (Figure 4A). mrna export activity is reduced by about 90% by a reverse charge mutation of Asp482 to Arg in TAP, which was constructed to abolish a direct salt bridge interaction with p15 Arg134 (Figures 1B and 4A). Substitution

7 FG Nucleoporin Recognition by TAP-p15 Heterodimer 651 Figure 4. Dissection of the Heterodimerization and NPC Binding Properties of TAP-p15 by Site-Directed Mutagenesis (A) Histogram showing the results of stimulation of mrna export activity using a CAT assay as described in the text. Human cells were transfected with either GFP alone or TAP alone as control experiments. In all other cases, cells were cotransfected with either TAP (wild-type or mutant) and p15, or with p15 and TAP mutants. The stimulation is expressed as the percentage of the activity of wild-type TAP-p15 heterodimers, and the error bars are calculated over four independent experiments. The Western blot analysis indicates that the expression levels of TAP mutants were comparable to that of the wild-type control. (B) Multidomain organization of human TAP-p15. The domain boundaries of the RNP and LRR domains (Liker et al., 2000), of the NTF2-like domain (present study), and of the predicted UBA-like domain (Suyama et al., 2000) are indicated. The NTF2-like domains of TAP and p15 are light and dark gray, respectively. The two black diamonds indicate the FG nucleoporin binding sites characterized in the NTF2-like domain (Leu383 and Leu386 from the present study) and mapped by mutagenesis in the UBA-like domain (Braun et al., 2001; Suyama et al., 2000). (C) Nuclear envelope association of TAP mutants in vivo. Hela cells were cotransfected with plasmid derivatives expressing p15 and TAP as fusion proteins. Approximately 20 hr after transfection, cells were extracted with Triton X-100 prior to fixation. TAP loses nuclear rim association when both FG binding sites in the NTF2-like and UBA-like domains are abrogated (L383,386R W594A mutant). On the other hand, p15 does not localize at the nuclear rim when unable to associate with TAP (R134D mutant), as discussed in the text. (D) Dominant-negative effect of a TAP mutant carrying mutations in the two FG nucleoporin binding sites. HeLa cells expressing the GFP- TAP mutant L383,386R W594A accumulate poly(a) RNA in the nucleus, as detected by oligo-dt in situ hybridization.

8 Molecular Cell 652 in 15% of normal export activity, while overexpression FG binding sites are mutated (TAP L383,386R W594A). of p15 alone is unable to promote export (Braun et al., The localization of p15 at the nuclear rim when coex- 2001). Strikingly, restoration of the salt bridge between pressed with the L383,386R W594A triple mutant is TAP residue 482 and p15 residue 134 by overexpressing likely due to the presence of endogenous TAP, since no the two proteins carrying reverse charge mutations (TAP rim staining is detected when overexpressing a mutant D482R and p15 R134D) restores the export activity to p15 (R134D) that is unable to interact with TAP (Figure about 75% of the wild-type value (Figure 4A). 4C). Strikingly, the p15 R134D mutant can be recruited In yeast, mutation of His400 to Tyr in the NTF2-like to the nuclear envelope by coexpressing the TAP mutant domain of Mex67p impairs the interaction with its part- D482R, which restores a critical salt bridge at the heterodimeric ner Mtr2p and association with nuclear pores, resulting interface. The immunofluorescence data are in in mislocalization of the protein to the cytoplasm at the agreement with the structural data, indicating that p15 nonpermissive temperature (Santos-Rosa et al., 1998; does not bind FG repeat nucleoporins directly, and that Segref et al., 1997). In human TAP, the equivalent residue its association to the NPC is mediated by TAP. (Asn496; Figure 2) is found in the heterodimeric interface near Arg134 of p15 (Figure 1B). This supports the notion Two FG Nucleoporin Binding Sites in the NTF2-like that human p15 and yeast Mtr2p might be functionally and UBA-like Domains Act Synergistically and structurally related despite the lack of obvious se- in TAP-Mediated mrna Export quence similarity (Katahira et al., 1999; Strä er et al., Modification of the nucleoporin binding properties of 2000; Suyama et al., 2000). While mutations of p15 at TAP is expected to affect its transport activity, as has the heterodimeric interface result in drastic effects on been reported for the import factors NTF2 and importin mrna export, mutations of conserved residues that are (Bayliss et al., 1999, 2000; Lane et al., 2000; Ribbeck exposed on the outer surface of p15 (Phe20, Asp29, and and Görlich, 2001). The contribution of the two nucleo- Arg31) result in no significant decrease in export activity porin binding sites in the NTF2-like and UBA-like do- (data not shown). The mutagenesis data suggest that mains of TAP to mrna export was analyzed by measurp15 mainly functions in stabilizing the NTF2-like domain ing the export activity of structure-based mutants with of TAP by heterodimerization, although other functions the CAT assay described above (Figure 4A). Disruption can not be ruled out (with the exception of NPC binding of the FG binding pocket of the TAP NTF2-like domain and Ran binding; see below). by the L383,386R double mutation results in a decrease of TAP activity by about 80% (Figure 4A). A less severe NPC Association of TAP-p15 Heterodimers Is disruption of the hydrophobic pocket by mutation of Mediated by Two FG Binding Domains of TAP Pro521 to Asn reduces the mrna export activity of TAP The crystallographic analysis shows that the NTF2-like by about 35%. In contrast to what was reported for domain of TAP functions in binding FG repeat nucleo- the NTF2 Asp23 to Ala mutation (Lane et al., 2000), the porins at a single hydrophobic pocket, and suggests corresponding mutation in either TAP (Asp399) or p15 that p15 contributes only indirectly by maintaining a (Asp29) does not increase the efficiency of the transport properly folded and functional NTF2-like scaffold. A second factor. Indeed, the TAP 399 mutant has a decreased FG nucleoporin binding site in TAP is known to be activity (probably due to local structural changes), since provided by the UBA-like domain (Figure 4B; Bachi et the mutated aspartic acid is involved in a structural inter- al., 2000; Herold et al., 2000; Suyama et al., 2000). To action with His474. study the contribution of the two FG binding sites to As in an earlier report (Braun et al., 2001), the W594A NPC association in vivo, we disrupted them by site- mutation in the UBA-like domain results in a decrease directed mutagenesis and investigated nuclear rim association. in mrna export activity by 88%, and a similar result is In the context of the new structural and bio- obtained by deleting the entire UBA-like domain (Figure chemical data, the immunofluorescence experiments 4A). The triple mutation of Leu383, Leu386, and Trp594 were performed in the presence of both TAP and p15 abolishes the mrna export activity completely (Figure to ensure the proper folding and function of the NTF2-4A). The activity data are in agreement with the immuno- like domain. TAP mutants fused to GFP were coex- fluorescence data (Figure 4C), and indicate that the two pressed with p15 fused to two IgG binding units of protein FG binding sites in the NTF2-like and UBA-like domains A from S. aureus (zz tag). Transfected HeLa cells of TAP are responsible for all the NPC association prop- were extracted with Triton X-100 prior to fixation. Most erties of the heterodimer. Remarkably, overexpression of the nucleoplasmic and cytoplasmic pools of the pro- of this triple mutant in HeLa cells inhibits the export of teins dissolved under these conditions. However, proteins bulk polyadenylated RNAs (poly[a] RNA) in a dominant- associated with the nuclear envelope resist extrac- negative manner (Figure 4D). tion and are clearly seen as a rim around the nucleus. In the case of TAP-mediated export of CTE-containing In the presence of zzp15, disruption of the FG binding RNAs, Xenopus oocyte experiments indicate that either pocket of the TAP NTF2-like domain (Figure 3A) by a NPC binding domain is sufficient for U6-CTE export double point mutation of Leu383 and Leu386 to Arg (data not shown). In the case of mrna export, each (L383,386R) diminishes the nuclear rim staining typical NPC binding site in the NTF2-like and UBA-like domains of wild-type GFP-TAP (Figure 4C). Similarly, nuclear rim contributes at most 20% to the TAP export activity, staining is diminished by substituting Trp594 to Ala in suggesting that the two domains act synergistically in the UBA-like domain, which has been previously shown NPC association and translocation of mrna cargoes. to affect FG binding in vitro (Bachi et al., 2000; Suyama Deletion of the UBA-like domain or abolition of its FG et al., 2000). Nuclear rim staining is abrogated when both binding properties has a similar effect on the mrna

9 FG Nucleoporin Recognition by TAP-p15 Heterodimer 653 Figure 5. RanGDP Binding Abilities of NTF2-like Domains (A) Structure of an NTF2 monomer (green) bound to RanGDP (blue; Stewart et al., 1998). In particular, the Phe72 residue from the switch II region of RanGDP (see enlargement) interacts with a pocket of NTF2 lined by hydrophobic residues (green; Stewart et al., 1998). (B) The structure of the NTF2-like domain of TAP is incompatible with a similar RanGDP binding due to the presence of the insertion loop. (C) The structure of p15 shows that certain residues are similarly positioned in the RanGDP binding pocket of NTF2 (see Trp47 and Phe101). However, the access of RanGDP is prevented by the occluding residues Phe135 and Arg107. export activity (mutants UBA and Trp594 in Figure 4A), nucleoporin binding pocket (Figure 5A). The pocket is implying that the role of the UBA-like domain is confined occluded in both TAP and p15. to NPC association. Disruption of the FG binding site in In the TAP structure, the occlusion is macroscopic the NTF2-like domain has a milder effect on mrna export and due to the insertion loop characteristic of this NTF2- than deletion of the entire domain, which effectively like molecule (Figure 5B). In particular, superposition of abolishes mrna export ( NTF2 and Leu mutant the TAP and NTF2-RanGDP structures shows that helix in Figure 4A). This suggests that the TAP-p15 NTF2-2A in the insertion loop of TAP would clash with the like scaffold might have an additional function in mrna switch II region of RanGDP. The occlusion is more subtle export other than NPC association, similar to the but still unambiguous in the case of p15 (Figure 5C). RanGDP import factor NTF2, which binds both nucleoporins Hydrophobic residues of p15 are present at identical and its RanGDP cargo. positions to those lining the NTF2 cavity, but the larger side chains fill the cavity. In particular, the position of TAP and p15 NTF2-like Structures Are Incompatible the incoming Ran switch II Phe72 is occupied by the with Ran Binding Phe135 of p15 and further restricted by the conserved Despite the overall similarity with the RanGDP export Arg107 and Asp137 (Figure 2). factor NTF2, in vitro binding studies have shown that Although it has been reported that p15 binds RanGTP TAP-p15 is unable to bind RanGDP (Katahira et al., 1999; (Black et al., 2001), other studies failed to detect the Herold et al., 2000). The interaction between RanGDP interaction (Herold et al., 2000; Katahira et al., 1999). and NTF2 is mediated mainly by the switch II region of The structural analysis shows that RanGTP cannot bind the GTPase, with a minor contribution from the switch the TAP-p15 heterodimer in a manner similar to that I region (Stewart et al., 1998). In particular, the aromatic observed in the RanGDP/NTF2 complex. The same disside chain of Phe72 protruding from the switch II region criminants against RanGDP binding would be accentuated of Ran binds to a hydrophobic pocket distinct from the in the case of RanGTP, where the conformation

10 Molecular Cell 654 of both switch I and switch II regions would not be residues , and TAP UBA corresponds to a deletion of compatible with these NTF2-like structures. The residues Mutations were introduced using an oligonucleo- tide-directed in vitro mutagenesis system from Stratagene (Quick- RanGTP switch II region would protrude further into the change site-directed mutagenesis). TAP mutants were generated in NTF2-like cavity and the switch I region would clash plasmids pgexcs-tap and pegfp-c1-tap. These plasmids allow with the loop connecting strands 4 and 5. TAP expression either as glutathione S-transferase (GST) fusion in E. coli or as a fusion with green fluorescent protein (GFP) in mammalian Conclusions cells, respectively. Mutants of p15 were generated in plasmid pegfp-n3zzp15-1 (Braun et al., 2001), which allows expression of p15 with an N-terminal tag consisting of two immunoglobulin binding The NTF2-like structural framework has evolved differdomains of protein A from S. aureus (zz tag) in mammalian cells. ent functions in nucleocytoplasmic transport. In NTF2, GST protein fusions were expressed in E. coli BL21(DE3) strains. For the scaffold binds the cargo RanGDP and binds oocyte injections, recombinant proteins were purified as previously nucleoporins efficiently enough to associate with the described (Grüter et al., 1998) and dialyzed against 1.5 PBS sup- nuclear rim and to mediate NPC translocation. In the plemented with 10% glycerol. TAP-p15 NTF2-like scaffold, relatively minor structural Protein Crystallization changes prevent binding to Ran (GDP- or GTP-bound), Human TAP (residues ) and p15 were coexpressed in E. coli which is neither a cargo nor a direct regulator of TAP- BL21(DE3) as GST fusion (ampicillin-resistant) and untagged (kanamediated mrna export. The NTF2-like domain of TAP mycin-resistant) proteins, respectively. After affinity purification on interacts specifically with FG repeat nucleoporins, and GST sepharose (Pharmacia), the protein was dialyzed in buffer A a similar FG binding pocket can be located in the struccleaved (50 mm Tris [ph 8.5], 50 mm NaCl, and 0.5 mm DTT) and the GST was ture of NTF2. However, the structure of p15 is incompatichromatography by overnight incubation with TEV protease. Anion exchange (Bio-Rad) at ph 8.5 allowed the separation of pure ble with FG repeat nucleoporin binding. This is unex- TAP-p15 complex from GST. pected, since p15 shares a higher degree of sequence The complex was crystallized by vapor diffusion at 18 C after similarity to NTF2 than does TAP, and suggests that the mixing the protein solution at 12 mg/ml in buffer A with an equal location of translocation-promoting properties on the volume of a well solution containing 0.8 M Na/K tartrate and 100 mm surface of transport factors is not easily predictable, MES (ph 6.4). Bipyramidal crystals grew within a week to a size of even when they display gross structural homology m. Crystals were cryoprotected with 30% glycerol added to the mother liquor and flash frozen in liquid nitrogen-cooled The regions of the NTF2-like class of transport factors propane. The crystals are hexagonal, in space group P giving rise to their translocation-promoting properties (a b Å, c Å) with one complex per asymmetric are structurally distinct from those of the importin- -like unit and having 60% solvent content. A native data set to 1.9 Å class. Nonetheless, the FG nucleoporin repeats assume resolution was collected at the ESRF beam line ID14-1, and pro- a similar conformation on binding both classes of trans- cessed with the Denzo/HKL package (Otwinowski and Minor, 1997). Cocrystals of TAP-p15 and an FG repeat-containing peptide were port factors. Despite the fact the FG binding pockets of obtained under the same conditions as described above. A peptide importin and of the NTF2-like domain are structurally with sequence GQSPGFGQGGSV (corresponding to the nucleoporin unrelated, in both cases the conformationally flexible CAN residues ) was chemically synthesized (MWG-Bioglycine of the nucleoporin repeat provides the flexibility tech) and purified by HPLC. The peptide was resuspended in 50 mm for the contiguous phenylalanine to reach the pocket. Tris (ph 7.5), 50 mm NaCl and the ph was readjusted to 7.5. The Flexibility would be all the more required in the context protein and peptide were mixed to a molar ratio of 1:40 in the presence of mother liquor. Cocrystals were grown over 10 days in of the multiple FG repeat-containing nucleoporins that sitting drops in the same form as the native. Data were collected line and crowd the NPC. at the ESRF beam line ID14-3 to a resolution of 2.8 Å. Data collection TAP-mediated translocation of mrna cargoes re- statistics are summarized in Table 1. quires the association of its NTF2-like heterodimeric domain and UBA-like domain with components of the Structure Determination and Refinement NPC. Each domain features a single FG nucleoporin The heavy atom derivatives were made by soaking for 20 hr in 50 M methyl mercury acetate. A three-wavelength MAD experiment was binding site, which acts synergistically in mrna transloperformed at the Elettra synchrotron (Trieste) around the mercury cation. The TAP homologs yeast Mex67p, human NXF2, absorption edge (Table 1). The SOLVE program (Terwilliger and and C. elegans NXF1 are likely to employ a similar mech- Berendzen, 1999) identified seven Hg sites with occupancies ranganism of NPC association. It is possible that the two ing from 0.4 to 0.7, giving experimental phases with an overall figure NPC binding domains might have different relative affini- of merit of 0.61 at 2.9 Å resolution. Solvent flattening using the ties for FG nucleoporins in different NXF homologs, thus Resolve program (Terwilliger, 2000) resulted in an interpretable elec- tron density map. providing a rational for the apparently discordant reports The model was built with O (Jones et al., 1991) and refined using in the literature on the contribution of the two domains the maximum likelihood target in the CNS program (Brünger et al., to nucleoporin binding. Finally, the essential function of 1998) against the 1.9 Å native data set. No cutoff was applied, the NTF2-like TAP-p15 heterodimer in mrna export is and a random sample of 4% of the data was excluded from the intriguing, as it might go beyond its FG binding properreochemistry, refinement for unbiased monitoring. The final model has good ste- ties. Whether, as in the case of the RanGDP import with only one well-ordered residue (Thr458 of TAP) lying in a disallowed region of the Ramachandran plot. This position factor NTF2, the NTF2-like TAP-p15 heterodimer conis occupied by a glycine in all other TAP homologs (Figure 2). tributes not only to NPC association but also to binding The TAP-p15-FG repeat peptide structure was solved using cellular cargoes (either by itself or in conjunction with phases from the TAP-p15 model. After rigid body refinement, the other domains) is an open question. R free dropped to 30.5%. A single cycle of rebuilding and refinement resulted in an R free of 26.9%. The refinement statistics are shown in Experimental Procedures Table 1. Plasmid Preparation and Expression of Recombinant Proteins Most of the plasmids used in this study have been described before (Braun et al., 1999, 2001). TAP NTF2 corresponds to a deletion of DNA Transfections, CAT Assays, and Immunofluorescence The CAT assays were performed essentially as described previously (Braun et al., 2001). Briefly, human 293 cells were transfected with

11 FG Nucleoporin Recognition by TAP-p15 Heterodimer 655 a mixture of plasmids encoding -gal, CAT (pcmv128), and either Calado, A., and Carmo-Fonseca, M. (2000). Localization of poly(a)- GFP alone or fused N-terminally to TAP or various TAP mutants. binding protein 2 (PABP2) in nucleus speckles is independent of For cotransfections, a pegfp-n3 derivative encoding zzp15 was import into the nucleus and requires binding to poly(a) RNA. J. Cell used. Cells were collected 48 hr after transfection and -galactosidase Sci. 113, and CAT activity were determined. Protein expression levels Carson, M. (1991). Ribbons 2.0. J. Appl. Crystallogr. 24, were analyzed by Western blot using anti-gfp antibodies. Chaillan-Huntington, C., Braslavsky, C.V., Kuhlmann, J., and Stew- To determine the subcellular localization of TAP mutants, HeLa art, M. (2000). Dissecting the interactions between NTF2, RanGDP, cells were transfected with pegfpn3zzp15 and pegfpc1 derivaand the nucleoporin XFXFG repeats. J. Biol. Chem. 275, tives encoding TAP and TAP mutants using FuGENE6 (Roche). Approximately 20 hr after transfection, cells were permeabilized with Conti, E., and Izaurralde, E. (2001). Nucleocytoplasmic transport 0.5% Triton X-100 for 1 min on ice and then fixed in formaldehyde. enters the atomic age. Curr. Opin. Cell. Biol., in press. The zz fusion proteins were visualized using a rabbit polyclonal anti- Evans, S.V. (1993). SETOR: hardware-lighted three-dimensional protein A antibody (Sigma; dilution 1:1000 in PBS supplemented solid model representations of macromolecules. J. Mol. Graph. 11, with 5% FCS and 0.05% Tween 20) and a secondary Cy3-coupled anti-rabbit IgG antibody (diluted 1:4000). Cover slips were mounted Görlich, D., and Kutay, U. (1999). Transport between the cell nucleus in VectaShield medium (Vector Labs). In situ hybridizations with and the cytoplasm. Annu. Rev. Cell Dev. Biol. 15, oligo-dt were performed essentially as described previously (Calado and Carmo-Fonseca, 2000). Grüter, P., Tabernero, C., von Kobbe, C., Schmitt, C., Saavedra, C., Bachi, A., Wilm, M., Felber, B.K., and Izaurralde, E. (1998). TAP, the Acknowledgments human homolog of Mex67p, mediates CTE-dependent RNA export from the nucleus. Mol. Cell 1, We are grateful to Michaela Rode for technical assistance, and thank Herold, A., Suyama, M., Rodrigues, J.P., Braun, I., Kutay, U., Carmo- Alberto Cassetta at the Elettra synchrotron (Trieste) for assistance Fonseca, M., Bork, P., and Izaurralde, E. (2000). TAP/NXF1 belongs with data collection, Mathias Wilm and Thomas Köcher (EMBL) for to a multigene family of putative RNA export factors with a con- mass spectrometry analysis, and Iain Mattaj, Peter Brick, and Elena served modular architecture. Mol. Cell. Biol. 20, Fernandez for useful comments on the manuscript. S.F. was sup- Holm, L., and Sander, C. (1993). Protein structure comparison by ported by an EMBO long-term fellowship (ALTF ). alignment of distance matrices. J. Mol. 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