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Nucleocytoplasmic transport is mediated by nuclear pore complexes (NPCs), enormous protein assemblies residing in circular openings in the nuclear envelope. The NPC is modular, with transient and stable components. The stable core is essentially built from two multiprotein complexes, the Y-shaped heptameric Nup84 complex and the Nic96 complex, arranged around an eightfold axis. We present the crystal structure of Nup1201-757, one of the two short arms of the Y-shaped Nup84 complex. The protein adopts a compact oval shape built around a novel bipartite α-helical domain intimately integrated with a β-propeller domain. The domain arrangement is substantially different from the Nup85•Seh1 complex, which forms the other short arm of the Y. With the data presented here we establish that all three branches of the Y-shaped Nup84 complex are tightly connected by helical interactions and that the β-propellers likely form interaction site(s) to neighboring complexes.
The main feature that distinguishes eukaryotes from prokaryotes is the confinement of the genetic material into a membrane-enveloped nucleus. Since gene transcription and mRNA processing occur inside the nucleus while protein translation is restricted to the cytoplasm, transport across the double-layered nuclear envelope (NE) is essential for cellular homeostasis. The exchange of all molecules, including ions, proteins, and RNAs is facilitated exclusively by nuclear pore complexes (NPCs) (D'Angelo and Hetzer, 2008; Lim et al., 2008; Tran and Wente, 2006; Weis, 2003). NPCs are large protein assemblies of 40-60 MDa that are embedded in the nuclear envelope and exhibit an 8-fold rotational symmetry around a central axis in addition to an imperfect two-fold symmetry across the plane of the NE (Beck et al., 2007; Stoffler et al., 2003). Composed of multiple copies of ~ 30 proteins, termed nucleoporins (nups), the NPC has an outer diameter of ~100 nm while the central channel measures ~40 nm in width. Transmembrane nups directly connect the NPC to the NE, while the phenylalanine-glycine (FG) repeat-containing Nups line the interior of the pore. These FG-filaments mediate nucleocytoplasmic transport of cargo molecules across the NE. FG-filament bearing nups are anchored to the NPC scaffold built from architectural nucleoporins arranged in two large multiprotein complexes that form a membrane-proximal layer. The scaffold structure is very stable and undergoes virtually no turnover in the quiescent cell (D'Angelo et al., 2009), while many other nucleoporins have variable dwell times at the NPC (Rabut et al., 2004). In consequence, the NPC is a highly modular structure (Schwartz, 2005). Understanding the structure of the NPC therefore depends upon elucidating its basic scaffold.
The two essential architectural building blocks of the NPC are the Nup84 subcomplex and the Nic96 subcomplex. The components of the Nic96 subcomplex likely include Nic96, Nup53/59, Nup157/170, Nup188 and Nup192 (yeast nomenclature), as inferred from co-immunoprecipations (co-IPs) (Alber et al., 2007; Hawryluk-Gara et al., 2008; Marelli et al., 1998; Onischenko et al., 2009) and yeast-two-hybrid screens (Wang et al., 2009; Yu et al., 2008). Judged by immunolabeling, the Nic96 subcomplex might form a central ring within the NPC sandwiched between peripheral rings formed by Nup84 subcomplexes (Alber et al., 2007). In comparison to the Nic96 subcomplex, the Nup84 subcomplex is substantially better understood. It has 7 universally conserved members (yeastNup84/humanNup107, yNup85/hNup75, yNup120/hNup160, Nup133, yNup145C/hNup96, Sec13, and Seh1) and three additional members (Nup37, Nup43, and ELYS/Mel-28) to date found mainly in metazoa (Cronshaw et al., 2002; Gillespie et al., 2007; Loiodice et al., 2004; Rasala et al., 2006). In the fungus Aspergillus nidulans, distant Nup37 and ELYS orthologs have been described recently (Liu et al., 2009). The heptameric core Nup84 complex assembles tightly as shown by co-IPs and in vitro assembly (Harel et al., 2003; Lutzmann et al., 2002; Siniossoglou et al., 2000; Walther et al., 2003). Negatively-stained electronmicrographs of the assembled Nup84 complex reveal a branched Y-shaped structure, with two short arms and a kinked stalk connected at a central hub (Lutzmann et al., 2002).
Crystallographic analysis of the Y-complex has progressed quickly. The kinked stalk ends with a flexibly attached β-propeller domain (Berke et al., 2004) at the N-terminus of Nup133 followed by an irregular C-terminal helical stack domain that connects end-to-end to Nup84 (Boehmer et al., 2008); Whittle, unpublished). The Nup84•Nup133 interface defines at least one kink in the stalk. The opposite end of Nup84 links to Nup145C (Brohawn et al., 2008). Nup145C•Sec13 (Hsia et al., 2007) resides proximal to the hub (Lutzmann et al., 2002). Nup85•Seh1 forms one of the two short arms of the Y-shaped complex (Brohawn et al., 2008; Debler et al., 2008). Nup84, Nup85, and Nup145C are structurally related (Brohawn et al., 2008), despite very low sequence conservation, as are the β-propeller proteins Seh1 and Sec13.
Nup120 is the last remaining Y-complex component without structural information. Here we report the 3.0 Å crystal structure of Nup120 (residues 1-757 of 1037), which reveals a compact and rigid structure composed of an N-terminal β-propeller domain tightly integrated into a novel bipartite α-helical domain. Our structure largely defines the second short arm of the Y-complex. Comparison with other members of the Y-complex, phylogenetic analysis, in vitro binding experiments, and in vivo localization data suggest a role for Nup120 consistent with our lattice-like model of the NPC.
After systematic C-terminal truncation, a stable fragment comprising most of Nup120 (residues 1-757 of 1037 total) from S. cerevisiae was recombinantly expressed in E. coli and purified. The protein is a monomer in solution (data not shown). Native protein readily crystallized and selenomethionine derivatized crystals were obtained after microseeding with native crystals. Though both crystal forms were optically identical, the selenomethionine crystals diffracted better and were used exclusively in structural analysis. The structure of Nup120 was solved with one molecule per asymmetric unit by single-wavelength anomalous dispersion (SAD) on very strong Se-Peak data (all 9 Se sites are well ordered). The model is complete except for 27 residues at the C-terminus and 7 flexible loops (out of 43 total loops) and was refined to Rwork / Rfree of 24.4 % / 29.9 % (Table 1).
Nup120 folds into a continuous, prolate disk with overall dimensions of 90 Å × 55 Å × 35 Å. One half of the structure is formed by an N-terminal β-propeller domain that is intimately connected to a compact central domain built from two closely packed α-helical segments (Figure 1). Overall, the structure is better resolved in the α-helical segment than the β-propeller, likely a result of a paucity of packing contacts involving the latter. The β-propeller of Nup120 contains 7 consecutive blades that fan out from a central axis. The blades are formed by a β-sheet of 4 consecutive antiparallel strands, labeled A-D. Blade 7 is built from the very N-terminus of the polypeptide chain forming strand 7D and joining strands 7A-C to close the propeller in a velcro-like closure commonly observed in β-propeller domains (Chaudhuri et al., 2008). Blade 1 is 5-stranded, with strand 7D extending to form the additional strand 1E before connecting to strand 1A (Figure 1E). Blade 3 is somewhat unusual in that the outermost strand 3D is only loosely connected to strand 3C with a hydrogen-bonding network hardly visible in our structure and the sequence could only be tentatively assigned for strand 3D residues 204-216.
The α-helical domain that forms the second half of the molecule is constructed in a unique discontinuous manner. In total the domain contains 15 helices, labeled α1-α15. The first 4 helices form a compact bundle and are inserted between blades 6 and 7 of the β-propeller. The remaining 11 helices are C-terminal to the β-propeller and pack tightly against the 4-helix bundle to form one compact entity. The arrangement of the helices is highly irregular. The most prominent feature of the domain are two long helices, α11 and α12, which pack against each other and form a central stalk, defining the long axis of the domain. Helices α5-α9 wrap up and around this element, with helices α6/α7 and α8/α9 arranged in two stacked braces oriented perpendicular to the stalk. Helices α1, and α13-α15 meander back down and around the other side to bury most of the hydrophobic stalk. The remaining surface area of the two central helices is closed by the 4-helix insertion bundle. Taken as a whole, the structure of Nup1201-757 consists of a bipartite helical domain that is interrupted by a β-propeller.
Other than a collection of spurious small contacts crystal packing is mainly achieved by a domain swap of the terminal helices α15 and α15′ exchanging between two neighboring molecules (Figure 2A). The interface measures 1355 Å2, is entirely hydrophobic and highly complementary (Figure 2B). Domain swaps are regularly found in crystals (Liu and Eisenberg, 2002) and, as stated above, we do not observe dimerization of Nup120 in solution. We cannot rule out the possibility that the interface is physiologically relevant; sterically the domain swap is conceivable in the context of the entire molecule including the C-terminal 280 residues omitted in our construct. It is however more likely that the exposed hydrophobic patch is artificially generated by the truncation of the domain, since we also do not observe particularly high sequence conservation within helix α15. We speculate that in vivo the patch likely accommodates one of the additional helices from the C-terminal domain, or alternatively, is involved in interaction with a neighboring molecule. Whether the C-terminal domain is rigidly or flexibly tethered to Nup1201-757 is an open question.
Overall, sequence conservation between Nup120 orthologs is weak as is typically observed in scaffold nucleoporins (Brohawn et al., 2008; Jeudy and Schwartz, 2007). Most of the better-conserved residues are buried in the hydrophobic core of the protein and are involved in maintaining the structural integrity of the protein. On the protein surface we find few conserved patches (Figure 3A). Most distinct is an area on the edge of the β-propeller, corresponding to the outer strands of blade 3 and the loop leading into blade 4. The conserved sequence begins in the 3BC loop and continues into strand 3C itself. Although generally buried in canonical β-propellers, here strand 3C is quite exposed. This is probably the result of weaker interactions with strand 3D, which is flanked by two large loops and peels away from the core of the propeller. Additional conserved residues are spotted around this area, creating a relatively large conserved patch. The potential significance of this observation is discussed below.
We analyzed the charge distribution on the surface of Nup120 (Figure 3B). Since Nup120 is part of the scaffold structure of the NPC, we asked whether it may be possible that it directly juxtaposes the pore membrane. This would also be consistent with a membrane-curvature sensing ALPS motif, predicted in helix α5-6 of Nup120 (Drin et al., 2007). The surface charge of Nup1201-757, however, is fairly mixed without conserved positive patches that might suggest direct membrane interaction. The ALPS motif is embedded in the structure and it is rather unlikely that it would swing out and insert in the membrane. Thus we suggest that Nup1201-757 does not directly touch the nuclear membrane.
Structure-guided sequence comparison of Nup120 and its human ortholog Nup160 strongly suggests that both proteins adopt the same unique fold despite a low sequence identity of ~10 %. Both non-canonical characteristics of Nup120 (the helical insertion between blades 6 and 7 of the N-terminal β-propeller and the long central stalk helices forming the hydrophobic core of the central domain) are clearly conserved in Nup160. The 279 additional residues of Nup160 are dispersed over several regions and mostly correspond to different loop lengths connecting α-helices and β-strands. Of note, the C-terminal domain of Nup160, which is not present in the Nup120 crystal structure described here, has 5 additional predicted helices, possibly indicating a vertebrate-specific extension. Despite these differences, the Nup120 crystal structure is likely generally representative of all Nup120/Nup160 orthologs.
We sought to map the interaction of Nup120 with its direct binding partners in the Y-complex, Nup145C and Nup85. In a gel filtration assay, we tested for the formation of a pentameric Sec13•Nup145C•Nup120•Nup85•Seh1 complex (Figure 4). Incubating Nup120766-1037 or Nup1201-757 with both Nup145C• Sec13 and Nup85•Seh1 resulted in complex formation only for the C-terminal Nup120 domain, but not for the crystal construct. In combination with previous interaction mapping experiments (Brohawn et al., 2008), we conclude that the helical tails of the ACE1 domains of both Nup145C and Nup85 each interact directly with the helical Nup120766-1037. This positions the C-terminus of Nup120 at the center of the hub of the Y-complex.
Having established that Nup120766-1037 is sufficient to bind both Nup145C• Sec13 and Nup85•Seh1 in vitro, we sought to examine the integration determinants of Nup120 into the NPC in vivo. NUP120 is not essential in yeast but nup120 Δ cells exhibit a pore clustering phenotype (Aitchison et al., 1995; Heath et al., 1995) that is reminiscent of but less severe than the pore clustering observed for other scaffold nucleoporins including Nup84 and Nup133 (Li et al., 1995; Pemberton et al., 1995; Siniossoglou et al., 1996). We genomically GFP-tagged full length Nup120 and replaced the C-terminal 280 residues of genomic Nup120 with an in frame GFP-tag to create strains expressing Nup120-GFP or Nup1201-757-GFP in a BY4741 background and examined the localization of the proteins via immunofluorescence (Figure 5). Nup120-GFP properly localizes to the NPC and shows typical nuclear rim staining, superimposing well with mAb414-staining of FG-Nups (Aris and Blobel, 1989). Nup1201-757-GFP, on the other hand, does not properly localize to the nuclear envelope and shows staining throughout the cell. This result is consistent with our in vitro data and suggests that the integration into the Y-complex is important for proper localization of Nup120.
A recent surge in the X-ray crystallographic analysis of components of the NPC has greatly increased the repertoire of available structures of nucleoporins constituting the structural scaffold of the NPC. These structures (including those of Nic96 (Jeudy and Schwartz, 2007; Schrader et al., 2008), Nup133-NTD (Berke et al., 2004), Nup133•Nup107 interaction complex (Boehmer et al., 2008), Nup145C•Sec13 (Hsia et al., 2007), and Nup85•Seh1 (Brohawn et al., 2008; Debler et al., 2008) as well as associated biochemical experiments, have led to a deeper and broader understanding of how the scaffold of the NPC is assembled from its constituent parts.
The structural subunits of the NPC were initially predicted to be composed of simple combinations of regular α-helical solenoids and β-propellers (Devos et al., 2006). Experimental data now allows to specify these broad classifications, which should help to more specifically address the ancestry of the NPC. Both Sec13 and Seh1 form open, 6-bladed propellers that are completed in trans by the N-terminal insertion blades of their binding partners Nup145C and Nup85, respectively. Furthermore, helical nucleoporins Nic96, Nup145C, Nup85, and Nup84 are built around a common and distinct ancestral coatomer element (ACE1) shared with Sec31 of the outer coat of COPII vesicles (Brohawn et al., 2008). In ACE1 proteins, a specific N-terminal elaboration is followed by a tripartite helical domain composed of a trunk, a crown and a tail element. The ~ 30 helices within ACE1 follow a J-like pattern, zig-zagging up on one side of the trunk, making a U-turn within the crown domain, and then following down on the opposite side of the trunk (Figure 6A, right panel). The tail domain is often attached with modest flexibility to the trunk and is missing in most crystal constructs. In the case of Nup145C and Nup85 the N-terminal elaborations are the aforementioned insertion blades that bind to Sec13 and Seh1. Nup145C•Sec13 and Nup85•Seh1 heterodimers form the two proximal segments of the Y-shaped complex and are tethered together by Nup120 (Brohawn et al., 2008).
Based on structure predictions and its overall size, it was reasonable to suggest that Nup120 may take on a structure similar to Nup145C•Sec13 and Nup85•Seh1, with the only major difference being that the β-propeller and the α-helical domains are fused into one polypeptide chain. However, comparison between the structure of Nup120 and the Nup85•Seh1 heterodimer reveals a marked difference in topology (Figure 6A). Whereas the ACE1 architecture of Nup85 forms an elongated α-helical domain, the central α-helical domain of Nup120 is nearly as wide as it is long, forming an almost globular structure. The ACE1 trunk module covers the bottom face of the Seh1 β-propeller, while in Nup120 the helical domain is attached to and integrated into an edge of the β-propeller. Further, the ACE1•β-propeller interaction is accomplished by the addition of an insertion blade N-terminal to ACE1, while in Nup120 the β-propeller domain inserts a 4-helix bundle into the central α-helical domain. This helical insertion fits snugly into a pocket formed by helices α5-α7 and α11-α13 and creating an interface of nearly 600 Å2 (Figure 6B).
The extensive interaction between the β-propeller and α-helical domain of Nup120 creates a large, rigid interface of 2175 Å2. In contrast, the largest contact area between ACE1 and its β-propeller partner is at the insertion blade/β-propeller interface. Additional contact areas in ACE1•β-propeller complexes are smaller in comparison to the corresponding interfaces in Nup120 and, importantly, far less hydrophobic. Thus, for the ACE1•β-propeller assembly one has to consider substantial flexibility about the interaction joint, while the Nup120 structure presented here is very likely inflexible. Not only does the structure of Nup120 significantly differ from the ACE1•β-propeller heterodimers, but additional emerging evidence suggests that it also lacks similarity to Nup170 and Nup133, the two other scaffolding nucleoporins of similar size and domain composition with an N-terminal β-propeller followed by an α-helical domain (Whittle & Schwartz, manuscript in preparation).
Here we report the crystal structure of Nup120, a large, universally conserved architectural nucleoporin. This structure adds substantially to the growing inventory of crystallographically characterized nucleoporins. As a result of these studies, we learn that the NPC is constructed from nucleoporins with a limited set of domain architectures. While other α-helical and β-propeller domains of scaffold nucleoporins fall into distinct classes, likely pointing to gene duplication in the early evolution of the NPC, the Nup120 architecture appears to be quite distinct. A search for structurally related proteins fails in detecting similarity beyond the isolated β-propeller scaffold or the arrangement of more than 6 α-helices. Within the list of crystallographically uncharacterized nucleoporins, none is likely to match the Nup120 structure closely.
Nup120 forms one of the two short arms of the universally conserved, 0.6 MDa Y-complex, the essential building block of the NPC scaffold. The assembly of the Y-complex from its 7 members is fairly well understood and has been studied using many different techniques. All of these studies profit from generally very high affinities observed between the interacting proteins within the Y, which generated largely consistent co-immunoprecipitation and yeast two-hybrid results and facilitated the crystallization of several complex crystal structures. While there is general agreement on the overall topology of the NPC, as determined by electron microscopic techniques, different models for the assembly of the NPC structural scaffold and the integration of the Y-complex are being discussed, as more detailed information is becoming available.
Based on a combination of computational, structural, biochemical, and in vivo experiments, a model was proposed where the Y-complex is positioned in two 8-membered rings located at the periphery of the NPC sandwiching two equally wide rings composed of Nup157/170,Nup188 and Nup192 in between (Alber et al., 2007). One exiting aspect of the combinatorial approach is, that going forward to a higher resolution it may allow the integration of crystallographic data as well, in which case it could come close to a detailed molecular description of the NPC.
Blobel and coworkers proposed a concentric cylinder model based on crystal-packing interactions where four 8-membered rings of the Y-complex are stacked and placed directly adjacent and in contact to the curved membrane (Debler et al., 2008; Hsia et al., 2007). Further, Nup85•Seh1 and Nup145C•Sec13 are both supposed to form heterooctameric fence poles spanning the NPC vertically, thereby connecting the 4 stacked rings. Nup157/170, Nup188, Nup192 and Nic96 are suggested to form a second inner layer bridging to a third layer composed of FG-nups. With a Y-complex scaffold twice the mass of the computer-generated model, the concentric cylinder model generates a densely packed NPC coat.
In contrast to the concentric cylinder model, we proposed a lattice-like model for the NPC, extrapolated from the assembly of COPII vesicle coats and substantiated by the structure and assembly principles of core components of the NPC scaffold (Brohawn et al., 2008; Brohawn and Schwartz, 2009). We propose the Y-complex does not directly coat the pore membrane (in analogy to the COPII outer coat), but is anchored by another set of proteins, likely involving the essential transmembrane nucleoporin Ndc1 and/or its direct binding partners (Onischenko et al., 2009). It is of interest to discuss this issue in respect to the membrane-inserting ALPS motif that was experimentally characterized within a loop structure in hNup133-NTD and that was predicted to occur as well in yNup85 and yNup120 (Drin et al., 2007). Based on the structural data now available on both yNup85 and yNup120, it appears unlikely that the predicted ALPS motif in both proteins is functional in membrane-binding since neither is in an exposed region of the protein, or is likely to become exposed. This is in contrast to the ALPS motif in hNup133-NTD, where it is well exposed in the crystal structure, and also highly conserved in metazoa (Berke et al., 2004). Taking all the available data together, it appears more reasonable to suggest a specific function for the ALPS motif in metazoan Nup133 rather than a general function in anchoring of the NPC to the pore membrane. Since Nup133-ALPS is only poorly conserved in yeast, it is tempting to speculate that it may have a specific role in NPC assembly in open mitosis (Güttinger et al., 2009). We predict that the lattice scaffold of the NPC is built from edge and vertex elements, following similar assembly principles as established for COPII. However, in the absence of definitive inter-subcomplex interaction data, any detailed NPC assembly model is still premature and has to be interpreted cautiously.
The fact that inter-subunit interactions are still obscure suggests that these interactions are rather weak and hard to establish. Each short arm of the Y-complex contains one β-propeller domain, while the stalk contains two (Figure 1). For the assembly of the extensions of the Y, direct interactions between the α-helical domains is essential, however this does not exclude the participation of the β-propellers. It is reasonable to suggest, that the β-propellers are prime candidates for the elusive inter-subcomplex contacts. The vertices of the outer coat of COPII vesicles are assembled exclusively via β-propeller interactions, which have still only been inferred by fitting crystal structures into EM maps (Fath et al., 2007; Stagg et al., 2008). β-propellers make excellent protein-protein interfaces due to their inherent ability to pair with a binding partner in multiple modes. Binding to peptides via the face of the β-propeller is well known (Jawad and Paoli, 2002). Additionally, each blade exposes on its edge (typically on strand D) a stretch of ~ 6-8 residues available for intermolecular β-sheet formation, which can be likened to one half of a zipper. In Nup120, 5 of the 7 blades are exposed this way, two are buried in the hydrophobic core shared with the attached α-helical domain. In addition to these interactions being relatively weak, another inherent difficulty in identifying them is that they are likely very poorly conserved at the sequence level because the contacts are mediated via the backbone rather than side chains. Based on the available data, it is conceivable that the Nup120 β-propeller is involved in inter-Y contacts. It is also possible that it is used to bridge to the Nic96 complex, but we can also not exclude that it may be an anchor for dynamic nucleoporins or other accessory proteins. The relatively mild nup120Δ phenotype (Figure 5) compared to nup133Δ or nup84Δ and the behavior of Nup1201-757-GFP suggests that if the Nup120 β-propeller has an integral role in the NPC scaffold, it is either redundant or can be functionally replaced by another nucleoporin.
In summary, we show that Nup120 adopts a unique architecture to build one of the two arms of the multimeric Y-shaped complex, the linchpin of the NPC scaffold. The atomic structure of the universally conserved heptameric core of the Y complex is now nearing completion. With reliable data on inter-subcomplex contacts the construction of a basic NPC architecture is within reach in the close future.
Nup120 from S. cerevisiae (residues 1-757 of 1037) was expressed at 18°C in E. coli strain BL21(DE3)-RIL as a 6xHis N-terminal fusion protein from a pET-Duet-derived plasmid. Cells were pelleted and resuspended in lysis buffer (50mM potassium phosphate pH 8.0, 500mM NaCl, 40mM imidazole, 5mM β-mercaptoethanol). Cells were lysed using a french press and the clear lysate incubated in batch with Ni-affinity resin. After washing the resin in batch with lysis buffer, the protein was eluted with lysis buffer containing 250mM imidazole. After cleavage of the purification tag, Nup120 was subjected to size exclusion chromatography on Superdex S200 equilibrated in 10mM Tris/HCl pH 8.0, 150mM NaCl, 0.1mM EDTA, and 1mM DTT. Nup120 eluted as a monomer of 88 kDa. Selenomethionine-derivatized protein was prepared as previously described (Brohawn et al., 2008) and Nup120-SeMet was purified identically to the native version.
Full length Nup85 in complex with Seh1 and a single chain version of full-length Nup145C in complex with Sec13 from S.cerevisiae were cloned as described (Brohawn et al., 2008), purified as for Nup120 (residues 1-757), and are referred to in the text as Nup85•Seh1 and Nup145C•Sec13. The C-terminal helical domain of Nup120 (residues 766-1037) was generated from a full length Nup120 construct by PCR. A 5-protein complex of Nup120 (residues 766-1037), Nup85•Seh1, and Nup145C•Sec13 was prepared by co-expression of a trimeric complex of Nup120 (residues 766-1037)•Nup85•Seh1 (Brohawn et al., 2008) and the single chain version of Nup145C•Sec13 in BL21(DE3)-(RIL) cells and was purified as for Nup120 (residues 1-757). The Ni-NTA elution was pooled, digested with human rhinovirus 3C to remove fusion tags, and subjected to size exclusion chromatography using a Superdex S200 26/60 column equilibrated in 10mM Tris/HCl pH 8.0, 250mM NaCl, 1mM DTT, and 0.1mM EDTA.
Nup120 concentrated to 20 mg/ml was crystallized in 15% (w/v) PEG 3350, and 0.1M Tris/HCl pH 7.5, 0.2M KSCN by the hanging drop vapor diffusion method at 18°C in 2μl drops. Crystals grew within 3-6 days forming rhomboid prisms with dimensions of 60μm × 60μm × 20μm. The selenomethionine derivative crystallized in the same condition, while the highest quality crystals were obtained by microseeding with native crystals. Both native and derivative crystals were cryo-protected by serial transfer of the crystals into reservoir solutions supplemented with increasing amounts of PEG200 (10%-25% (v/v), 5% steps) before flash freezing in liquid nitrogen. Both native and derivative protein crystallized in space group P21212 with one molecule per asymmetric unit. Data was collected at beamline 24ID-C at Argonne National Laboratory.
Although the native crystals were larger and optically superior, the selenomethionine-derivatized crystals diffracted significantly better and were exclusively used for data analysis. A complete dataset was collected at the Se-Peak wavelength and data reduction was carried out using the HKL2000 package (Otwinowski and Minor, 1997). All 9 selenium sites were found using SHELXD (Sheldrick, 2008). After refinement of the Se positions and density modification with SHARP, an adequate experimental electron density map was obtained, allowing for the assignment and building of the majority of the structure. Sequence assignment was aided by using the selenium positions as markers. Model building was done with Coot (Emsley and Cowtan, 2004) and refinement was carried out using the PHENIX suite (Adams et al., 2002). The model is complete except for residues 31-52, 188-200, 303-313, and 731-757 for which only spurious electron density was observed. Blades 3 and 4 of the β-propeller have the highest temperature factors and are not as well packed as the remainder of the molecule. Sequence assignment in this region, particularly in strand 3D residues 204-216, is tentative.
For Nup120 (residues 1-757), Nup145C•Sec13, and Nup85•Seh1 binding experiments, equimolar amounts of each component were incubated alone or in combination for 30 minutes at 4°C in binding buffer (10mM Tris/HCl pH 8.0, 250mM NaCl, 1mM DTT, 0.1mM EDTA). Reactions were injected onto a Superdex S200 hr10/300 column (GE Healthcare) equilibrated in binding buffer, and run at a flow rate of 0.8 ml/min (Figure 4A).
Deletion strains were taken from the Yeast Deletion Consortium (Winzeler et al., 1999), C-terminal GFP-tagging was done by homologous recombination in a BY4741 background, using pFA6a-GFP(S65T)-kanMX6 as template for C-terminal modifications (Longtine et al., 1998). Strains were selected on G418 plates (200 μg/ml) and verified by PCR.
Strains were grown overnight in YPD (1% yeast extract, 2% yeast peptone, 2% glucose) at 30°C, diluted 20-fold into fresh YPD, and grown for 4-5 hours at 30°C to OD600 ~0.5. Cells were harvested by centrifugation, fixed for 3 minutes in 3.7% formaldehyde/0.1M potassium phosphate pH 6.5, and prepared for immunofluorescence as previously described (Kilmartin and Adams, 1984). Samples were incubated with mAb414 (abcam, 1:1000) alone or in combination with goat anti-GFP (1:500) for 90 minutes at room temperature. Bound antibodies were detected by incubation with Cy5-conjugated anti-mouse (Jackson Labs, 1:500) alone or in combination with Cy2-conjugated donkey anti-goat (Jackson Labs, 1:200) for 45 minutes at room temperature. DNA was stained with 0.05 μg/ml 4′,6-diamidino-2-phenylindole (Sigma-Aldrich) and samples were mounted for imaging in 1 mg/ml p-phenylenediamine and 90% glycerol. Fluorescence microscopy was performed on a Zeiss AxioImager.Z1 microscope and images were taken with a Zeiss AxioCam HRm camera.
We thank staff at the NE-CAT beamline 24 at Argonne National Laboratory for excellent assistance with data collection; Iain Cheeseman for providing antibodies; Eric Spear for advice on in vivo experiments; Dennis Kim for use of his fluorescence microscope. Supported by NIH Grant GM77537 (T.U.S.), a Pew Scholar Award (T.U.S.), a Koch Fellowship Award (S.G.B.) and a Vertex Scholarship (S.G.B.).
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The atomic coordinates of the Nup120 structure have been deposited in the Protein Data Bank (PDB) with accession number 3HXR.