PMC:2728203 / 8193-22166 JSONTXT

{"target":"https://pubannotation.org/docs/sourcedb/PMC/sourceid/2728203","sourcedb":"PMC","sourceid":"2728203","source_url":"https://www.ncbi.nlm.nih.gov/pmc/2728203","text":"Results and Discussion\n\nThe Nab2 N-terminal domain binds to Mlp1\nPrevious work21 showed that full-length Nab2 binds to the C-terminal globular domain of Mlp1 (CT-Mlp1). We utilized the yeast two-hybrid system to identify the domain of Nab2 that interacts with CT-Mlp1. Nab2 can be divided into four domains,23 and hence, we constructed a series of two-hybrid bait plasmids expressing Nab2 in which each of the individual domains had been deleted (Fig. 1a). We used a lacZ reporter that generates a blue color when an interaction occurs.29 Each Nab2 plasmid was coexpressed with the vector alone (pJG4–5) or CT-Mlp1 (Fig. 1a). As shown in Fig. 1a, the negative vector controls (pEG202 and pJG4–5) were white, confirming that they did not activate the lacZ reporter. In contrast, coexpression of CT-Mlp1 and Nab2 generated the blue color, indicating that the lacZ reporter was activated, consistent with the proteins interacting with one another. CT-Mlp1 failed to interact with Nab2 only when the N-terminus was deleted (ΔN-Nab2), and the interaction was maintained with all other Nab2 fusion proteins. To determine if the N-terminal domain of Nab2 was sufficient for interaction with CT-Mlp1, we attempted to create a minimal yeast two-hybrid construct expressing only the N-terminal domain of Nab2. Unfortunately, this N-terminal domain fusion protein autoactivated the lacZ reporter; thus, we instead created a fusion consisting of both the N-terminal domain and the QQQP domain (Nab2-NQ). This protein interacted with CT-Mlp1 (Fig. 1a). Gfd1 was used as a positive control for interaction with the N-terminal domain of Nab2,27 and, as expected, Gfd1 interacted with the NQ domain but not with ΔN-Nab2 (Fig. 1a). For all two-hybrid experiments, expression of each fusion protein was confirmed by immunoblotting (data not shown). Overall, the two-hybrid data suggest strongly that the N-terminal domain of Nab2 is both necessary and sufficient to interact with the Mlp1 C-terminal domain.\nPull-down assays complemented the results obtained using the two-hybrid system and confirmed that the N-terminal domain of Nab2 without the QQQP domain is both necessary and sufficient to mediate interactions with Mlp1. A glutathione S-transferase (GST) fusion to the N-terminal domain of Nab2 (GST-Nab2-N, residues 1–97 of Nab2) was engineered, expressed in Escherichia coli, and purified. Either GST-Nab2-N or GST alone as a control protein was incubated in yeast lysate, and then, the GST protein and any associated proteins were bound to glutathione beads. As shown in Fig. 1b, a band of approximately 220 kDa that copurified with GST-Nab2-N but not with GST alone was visualized using Coomassie Blue staining. Immunoblotting confirmed that this band corresponded to the full-length Mlp1 protein (Fig. 1c). When the yeast lysate was prepared from mlp1Δ cells, this 220-kDa band was not detected by either Coomassie staining (Fig. 1b) or immunoblotting (Fig. 1c). Although the amount of Mlp1 copurified with GST-Nab2-N from yeast lysate was rather small, it is quite remarkable that a band corresponding to this full-length, very large, nuclear pore-associated protein17 can be isolated from yeast extract in a single step. The results of this experiment indicate that the N-terminal domain of Nab2 is sufficient to copurify Mlp1 from a complex mixture, showing that the interaction is highly specific.\nAn in vitro binding assay established that the N-terminal domain of Nab2 interacted directly with the Mlp1 C-terminus. Recombinant proteins were expressed and purified from E. coli as described in Materials and Methods. Either GST-Nab2-N or GST control protein was incubated with the Mlp1 C-terminal domain fragment. As shown in Fig. 1d, the Mlp1 fragment bound to GST-Nab2-N but not to the control GST protein, confirming that the N-terminal domain of Nab2 interacts directly with the C-terminal domain of Mlp1. The amount of binding between Nab2-N and CT-Mlp1 appears to be substoichiometric, which is consistent with the interaction between Nab2 and Mlp1 being relatively weak, to facilitate export rather than retention at the nuclear pore. Preliminary binding studies indicated that the Kd for this interaction is in the micromolar range (data not shown).\n\nStructure of the Nab2 N-terminal domain\nThe structure of the N-terminal domain of Nab2 was established using both NMR and X-ray crystallography. A fragment corresponding to residues 1–105 of Nab2 was produced in large quantities by bacterial expression and purified using conventional ion-exchange and gel-filtration chromatographic methods (see Materials and Methods). This material was exceptionally soluble, which facilitated the collection of NMR spectra, from which a model was generated using conventional methods. The final ensemble of 45 calculated structures of the Nab2 N-terminal domain is shown in Fig. 2a. Of the total of 50 structures calculated in the final round, these 45 correspond to a well-defined plateau region in the energy and energy-ordered root-mean-square deviation (rmsd) profiles (Fig. 2a), indicating that they form a suitable set for reporting structural statistics (Table 1).31 Residues Met1–Gln3 and Gly100–Ala105 were unstructured in solution, as evidenced by the lack of any medium- or long-range nuclear Overhauser enhancement (NOE) connectivities for these residues and by the sharpness and near random-coil chemical shift values of their NMR resonances. The remainder of the domain was well ordered, although there was slightly increased disorder in the loops, especially those between helix 1 and helix 2 (Pro22–Asp27) and, to a lesser extent, between helix 3 and helix 4 (Asp57–Ser60). The backbone rmsd over residues 4–99 was 0.47 ± 0.13 Å.\nAs illustrated in Fig. 2b, the solution structure of the N-terminal domain of Nab2 was based on a bundle of five α-helices (H1–H5). Unlike the highly compact bundle formed by helices H1–H4, each of which made multiple contacts with at least two of the other helices, helix H5 was less tightly associated with the rest of the structure. The only NOE constraints from helix H5 to other parts of the structure were all contacts to helix H1 (Ala84 contacts Asn9, Val12, and Ile13; Ile87 contacts Ile13 and Glu16; Ile91 contacts Glu16 and Ala19; Asn95 contacts Ala19 and Gly20). These contacts were sufficient to constrain the position of helix H5 with comparable precision to those of the other helices, but they were much fewer in number than the constraints involving any of the other helices. Further evidence for the relatively loose attachment of helix H5 to the rest of the structure came from the observation that NMR samples were slowly proteolyzed from the C-terminus. Over a period of several weeks, many signals assigned to residues of helix H5 were progressively lost from the spectra, whereas the signals assigned to helices H1–H4 were largely unaffected. The signals for protons in helix H5 were generally weaker than other components of the structure and decreased with time, consistent with its being gradually proteolyzed.\nWe also obtained P43212 symmetry crystals of the Nab2 N-terminal domain that diffracted past 1.8 Å resolution in-house (Table 2). We used the solution NMR structure of this domain as a model for molecular replacement and, after trying a number of different models containing different fragments of the structure, were able to obtain a unique solution using residues 7–82 (corresponding to helices H1–H4). After solvent flattening, the electron density enabled residues 6–94 to be built to produce a structural model that had an R-factor of 20.7% (Rfree = 26.4%) and excellent geometry (Table 2) after iterative cycles of refinement and rebuilding (Fig. 3).\n\nComparison between solution and crystal structures\nThe crystal and solution structures of the N-terminal domain of Nab2 were very similar for helices H1–H4 and superimposed with a Cα rmsd of 1.2 Å. However, the position of helix H5 varied significantly between the two structures as a result of a rigid-body rotation of the order of 25° (Fig. 4). This difference in the position of helix H5 could have been the result of crystal packing or might have been a consequence of partial proteolysis as it was only possible to trace the chain in the crystal structure as far as residue 94, whereas in the solution structure, this helix extended to residue 99. However, these differences are also consistent with the helix being only loosely associated with the rest of the structure, which is also consistent with the relatively few NOE connectivities to helices H1–H4 in the NMR data. Moreover, when the TLS (translation/libration/screw) rigid-body motion32 of the Nab2 N-terminal domain was modeled by assigning helices H1–H4 to one rigid body and helix H5 to another, the R-factor and Rfree derived from X-ray crystallography dropped substantially to 19.5% (from 20.7%) and 25.0% (from 26.4%), respectively. Analysis of the TLS motion using the TLDMD method33 indicated that the libration motions of helix H5 (20.1°2) were substantially greater than those of the helix H1–H4 core (libration of 4.6°2), consistent with helix H5 being substantially more mobile. Potentially, the movement of helix H5 might have a regulatory role in modulating the interactions between the Nab2 N-terminal domain and other components of the mRNA export machinery.\n\nSimilarity to the PWI fold\nA search for structural homologues of the Nab2 N-terminal domain using the DALI web site† indicated that it had distant similarity to the PWI domain fold (Fig. 4) that is found in a number of nucleic acid binding proteins such as the splicing factor and exon junction component SRm160.30,34 The Pro-Trp-Ile sequence for which the PWI domain is named is found in helix H1 of this fold and is important in generating the hydrophobic core of the PWI fold in which the Trp and Ile side chains pack against Phe101 in helix H4 of SRm160. The PWI fold is thought to bind both single- and double-stranded DNA and RNA through a characteristic basic patch on its surface.34 However, there is little conservation at the sequence level, and the characteristic Pro-Trp-Ile (PWI) sequence motif is not present in the Nab2 N-terminal domain where the corresponding residues are Val12, Ile13, and Val14. Similarly, Nab2 lacks the characteristic positively charged surface patch that has been associated with nucleic acid binding.34 Moreover, the nucleic acid binding function of SRm160 also requires residues upstream of the PWI domain34 that are lacking in the Nab2 N-terminal domain, and, consistent with this observation, we were unable to detect any interaction between the Nab2 N-terminal domain and poly(N) RNA in band-shift assays under conditions where the PWI domain from SRm160 showed a clear interaction (Fig. 5).\n\nPutative protein binding site centered on Phe73\nInspection of the solution and crystal structures of the Nab2 N-terminal domain showed the presence of a hydrophobic patch centered on Phe72 and Phe73 (Fig. 6a), suggesting that this region of the surface might represent a putative protein:protein interaction interface. Phe73 in particular was completely exposed on the surface of the protein. We therefore engineered a series of amino acid changes in these positions and assayed these mutants for binding to Mlp1 and Gfd1. Mutation of Phe72 had a negligible effect on the binding of the Nab2 N-terminal domain to either the Mlp1 C-terminal fragment or the Gfd1 C-terminus (Fig. 6b–d). In contrast, mutation of Phe73 to Asp or Ala dramatically reduced the affinity of Nab2 for the Mlp1 C-terminal fragment, whereas mutation to Trp did not decrease its affinity (Fig. 6b). Significantly, these mutations did not alter the binding of the Nab2 N-terminal domain to the Gfd1 C-terminal fragment, consistent with the mutations not altering the overall conformation of the domain (Fig. 6c). Binding experiments using yeast lysates confirmed that mutations in the hydrophobic patch on Nab2 interfered with the interaction between Nab2-N and the intact full-length Mlp1 protein. Equal amounts of GST-Nab2-N variants (WT, F72D, and F73D) were incubated in yeast lysate prepared from cells expressing protein-A-tagged Mlp1, and then, proteins associated with GST-Nab2-N were recovered using glutathione beads. Mlp1 that copurified with GST-Nab2-N was detected by immunoblotting for the protein A tag. As shown in Fig. 6d, full-length Mlp1 copurified with both wild-type Nab2-N and F72D Nab2-N. However, copurification of Mlp1 was not detected with either the GST control or the Nab2-N F73D mutant. Overall, the mutagenesis results are consistent with Phe73 forming a crucial component of the interaction interface between Nab2 and Mlp1 and hydrophobic interactions making an important contribution to the interface.\nIn summary, our results show that the N-terminal domain of Nab2 binds to the C-terminal domain of Mlp1 and that the Mlp1 binding site on Nab2 does not appear to overlap with the Gfd1 binding site. The Nab2 N-terminal domain has a fold based on a five-helix bundle that is analogous to the PWI fold found in SRm160 and other nucleic acid binding proteins, although it does not retain the nucleic acid binding function seen in other PWI domains. The hydrophobic side chain of Phe73 in the Nab2 N-terminal domain is exposed on the surface of the molecule and appears to be a crucial component of the Mlp1 binding site, although, clearly, further work will be required to define the precise nature of the interface between these two molecules. Our results are consistent with a model that envisages that interaction with Mlp proteins occurs soon before mRNA is exported and that Nab2 is important either for targeting the mRNA to the Mlp proteins or for releasing the mature mRNA from the Mlp proteins to enable its transit through the NPC. 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