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, Elena Conti  Molecular Cell  Volume 8, Issue 3, Pages 645-656 (September 2001) DOI: 10.1016/S1097-2765(01)00348-3

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 β 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 β 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 β 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 Molecular Cell 2001 8, 645-656DOI: (10.1016/S1097-2765(01)00348-3)

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 (α helices) and arrows (β strands). The stars indicate residues of p15 and TAP that are structurally equivalent to NTF2 (i.e., their Cα 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 Molecular Cell 2001 8, 645-656DOI: (10.1016/S1097-2765(01)00348-3)

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 3σ. 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 β 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 β (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 β, and corresponds to the structure shown in (E) Molecular Cell 2001 8, 645-656DOI: (10.1016/S1097-2765(01)00348-3)

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 Molecular Cell 2001 8, 645-656DOI: (10.1016/S1097-2765(01)00348-3)

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 Molecular Cell 2001 8, 645-656DOI: (10.1016/S1097-2765(01)00348-3)