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Correspondence to Günter Blobel: blobel{at}rockefeller.edu
Abstract
Nucleocytoplasmic transport occurs through nuclear pore complexes (NPCs) whose complex architecture is generated from a set of only 30 proteins, termed nucleoporins. Here, we explore the domain structure of Nup133, a nucleoporin in a conserved NPC subcomplex that is crucial for NPC biogenesis and is believed to form part of the NPC scaffold. We show that human Nup133 contains two domains: a COOH-terminal domain responsible for its interaction with its subcomplex through Nup107; and an NH2-terminal domain whose crystal structure reveals a seven-bladed ß-propeller. The surface properties and conservation of the Nup133 ß-propeller suggest it may mediate multiple interactions with other proteins. Other ß-propellers are predicted in a third of all nucleoporins. These and several other repeat-based motifs appear to be major elements of nucleoporins, indicating a level of structural repetition that may conceptually simplify the assembly and disassembly of this huge protein complex.
Abbreviations used in this paper: CTD, COOH-terminal domain; NE, nuclear envelope; NPC, nuclear pore complex; NTD, NH2-terminal domain.
Introduction
Macromolecular exchange between the cytoplasm and nucleus is a vital process involving a mobile phase of transport proteins and regulatory factors, and a stationary phase comprised of nuclear pore complexes (NPCs) embedded in the nuclear envelope (NE). Structural studies of mobile phase components have revealed the molecular details of cargo binding, regulation by Ran GTPase, and how transport factors interact with the NPC (Chook and Blobel, 2001), but the enormity of the stationary phase of nucleocytoplasmic transport has so far frustrated such efforts. Nonetheless, a low-resolution picture of the NPC and its organization is emerging. NPCs have a conserved eightfold symmetric framework with peripheral fiberlike extensions into both the cytoplasm and nucleus (Suntharalingam and Wente, 2003). An NPC has an estimated mass of 125 MD in vertebrates and 5572 MD in yeast, yet is comprised of only 30 proteins termed nucleoporins (Rout et al., 2000; Cronshaw et al., 2002). Nucleoporins are organized in subcomplexes that can be isolated from mitotic extracts or through biochemical extraction of the NE. These modular units are present in multiple copies arranged around two- and eightfold axes of symmetry and are believed to generate discrete structures within the NPC (Suntharalingam and Wente, 2003).
The nonameric Nup107-160 subcomplex in vertebrates (Loiodice et al., 2004) forms part of the peripheral circular core structure of the NPC and is located in close vicinity to the sharp bend between the outer and inner nuclear membranes (Belgareh et al., 2001). Immunodepletion of this subcomplex from Xenopus laevis egg extracts prevents reformation of even partial NPCs in nuclear reconstitution assays (Harel et al., 2003; Walther et al., 2003). Targeted depletion of Nup107 by RNA interference prevents integration of the subcomplex member Nup133, but allows other proteins of the subcomplex to be incorporated. Nup107-depleted NPCs were slightly compromised in their ability to export mRNA but did not affect the overall growth rate of cells (Boehmer et al., 2003; Galy et al., 2003; see, however, Walther et al., 2003).
In yeast, the homologous heptameric Nup84 subcomplex has been assembled in vitro from recombinant dimers and trimers produced in Escherichia coli (Lutzmann et al., 2002). By negative staining electron microscopy, the subcomplex has a Y-shaped structure with Nup133 located at the base of the stalk and Nup84 (the yeast homologue of Nup107) being its nearest neighbor. Nup133 depletion in yeast causes temperature-sensitive growth and mRNA export defects and clustering of NPCs at one pole of the NE (Doye et al., 1994; Pemberton et al., 1995).
Although an essentially complete inventory of nucleoporins is at hand and their organization into subcomplexes established, little information is available about the structural details of NPC architecture. Except for a 160-residue COOH-terminal fragment of Nup98 (Hodel et al., 2002), there is no other atomic structure of a nucleoporin available. Nup98 has been proposed to be a "mobile" nucleoporin from studies with GFP-tagged protein and FRAP experiments (Griffis et al., 2002). In contrast, members of the Nup107-160 subcomplex are stably associated with the NPC during interphase (Belgareh et al., 2001). Given the importance of this subcomplex in NPC assembly, its stability and its Y-shaped structure, the Nup107-160 subcomplex and its homologues have been proposed to form a portion of the central scaffold of the NPC (Belgareh et al., 2001; Harel et al., 2003). Here, we show the subcomplex member Nup133 contains two domains: a COOH-terminal domain (CTD) that anchors Nup133 via Nup107 to its subcomplex, and an NH2-terminal domain (NTD) that folds into a seven-bladed ß-propeller structure determined crystallographically at 2.35 Å. The discovery of a ß-propeller domain unexpected by sequence analysis prompted us to examine other nucleoporins for this fold. Candidate ß-propeller domains were found in three nucleoporins in addition to the six previously identified by their sequence repeats (Table I). High symmetry, modular subcomplexes built from a small component set, and high frequency of some structural modules show that the high degree of complexity in NPC organization is generated from multiple levels of modularity.
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Results and discussion
Nup133 contains two structural domains
Examination of the 1,156-residue human Nup133 by secondary structure prediction, disordered region prediction, and sequence conservation among homologues identified two domains: an NH2-terminal /ß domain of
400 residues and an all helical CTD of
640 residues (Fig. 1 a). Reconstitution of recombinant yeast Nup84 subcomplex revealed that Nup133 is anchored to the subcomplex via its direct interaction with Nup84, the yeast homologue of Nup107 (Lutzmann et al., 2002). Accordingly, Nup133 failed to assemble into NPCs of vertebrate cells depleted of Nup107 (Boehmer et al., 2003). In vitro binding experiments, performed with recombinant GST-Nup107 and in vitro transcribed and translated Nup133 proteins, show that the Nup133 CTD binds Nup107 (Fig. 1 b). GFP-tagged hNup133 (5021156) shows punctate nuclear rim staining consistent with integration into the Nup107-160 subcomplex and the NPC (Fig. 1 c).
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Structure of the Nup133 NTD reveals a ß-propeller fold
We expressed the NTD of hNup133 (residues 67514) in E. coli, crystallized it, and solved the structure to 2.35 Å. hNup133 NTD is a ß-propeller with seven, four-stranded ß-sheets arranged face to face around a central water-filled cavity (Fig. 2, a and b; and Video 1, available at http://www.jcb.org/cgi/content/full/jcb.200408109/DC1). The polypeptide chain enters each propeller blade from the innermost strand and folds in an antiparallel manner (Fig. 2 a and Fig. 3 a, strands labeled AD). Blade 7 of the propeller consists of the innermost three strands from the COOH terminus with the blade completed by the NH2 terminus of the domain. This 3 + 1 molecular clasp architecture is a common feature for stabilizing ß-propellers (Paoli, 2001). The repeating antiparallel structure results in a top surface composed of the loops connecting strand D of one blade to strand A of the next (DA loop) as well as the BC loop within each blade, whereas the bottom surface is composed of the AB and CD loops. Two significant -helical insertions are present in DA loops (Fig. 2, a and b, pink). The
1 helix inserts between the interface of blades 7 and 1, displacing blade 7 away and blade 1 toward the central axis. Blade 5 is extended and curls around helix
2 located in the DA loop connecting blades 4 and 5. This helical "wing" juts out from the core of the propeller by 15 Å. A disordered 20-residue insertion is present in the DA loop connecting blades 3 and 4 (DA34). The propeller has an overall diameter of 4550 Å and the ß-sheet core is
25-Å thick. Blade 2 has a short 310 helix just before strand 2A that projects into the top of the inner channel (Fig. 2 a; and Fig. 3 a, orange) and strand 2A is oriented away from the pseudo-sevenfold axis such that the bottom of the channel is wider than the top. The inner channel is oval shaped, being 1216 Å wide at the top and 1220 Å at the bottom (C
to C
), and contains many ordered water molecules.
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Conserved features suggest multiple interactions and a regulatable interaction motif
Proteins with the ß-propeller fold have diverse functions ranging from catalysis, intra- and extracellular signaling, vesicular sorting, and DNA binding (Paoli, 2001). The wide range of functional possibilities for this fold reflects the variety of interaction surfaces in the ß-propeller scaffold: top and bottom surfaces are composed of variable loops that can serve as a docking platform for other proteins; the side surface is composed of grooves at the ß-sheet interfaces often involved in peptide interactions; and the inner cavity potentially provides a space for sequestering ligands from bulk solvent. The lack of structural constraints on the evolution of a ß-propeller's primary sequence makes this an extremely adaptable module. Mapping the conservation of Nup133 NTDs from six vertebrate species, two insects, and two worms on the hNup133 propeller surface reveals conserved patches that extend along its circumference from blade 5 through blade 2 (Fig. 4 a, left and middle; and Fig. S1, alignment, available at http://www.jcb.org/cgi/content/full/jcb.200408109/DC1). The interface between blade 5 and 2-3 forms a conserved groove that is flanked at either end by negative charges (Fig. 4, a and b). The strongest surface conservation is centered on the
1 insert and a conserved hydrophobic groove runs between
1 and blade 7 (Fig. 4 a). Rotating around the pseudo-sevenfold axis of the propeller, a long disordered but conserved loop (DA34) follows strand 3D (Fig. 4 a, tube representation; and Fig. 4 c, sequence alignment). The loop lies above the entrance to a pocket in the interface between blades 3 and 4 (Fig. 4 b, right).
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Modularity of the NPC
NPC organization seems to be simplified at several structural levels. At the multiprotein level, a high degree of symmetry reduces the number of proteins required to generate such a large complex. In addition, these proteins are organized into modular subcomplexes, simplifying the number of interactions that need to be regulated for NPC assembly and disassembly to just those that mediate inter-subcomplex association. The structural modularity of Nup133 and the unexpected finding of a ß-propeller domain in it led us to examine other nucleoporins. Fold recognition algorithms predict ß-propellers in nine additional nucleoporins (Table I), making this domain a common module of the NPC. Systems specific to eukaryotes have been shown to be enriched in proteins derived from repeating elements (Marcotte et al., 1999), and other repeat modules are predicted or known to exist in the NPC including coiled-coils (Bailer et al., 2001), helical-solenoidforming repeats (Devos et al., 2004), and phenylalanine-glycine sequence repeats. Each class of repeat provides a scaffold with advantages for different modes of interaction (Andrade et al., 2001): helical solenoids have a large solvent-accessible surface well suited for large or tandem protein interfaces; coiled-coils are excellent mediators of oligomerization; compact, stable repeat structures such as ß-propellers are ideal for forming reversible interactions with several partners; and phenylalanine-glycine repeats provide disordered peptides for low-affinity, high-specificity interactions with karyopherins. Before the proteomic analysis of the yeast and vertebrate NPCs, it was estimated that at least 100 nucleoporins existed. In much the same way as the true component set was found to be simplified, perhaps the set of structural motifs within the NPC is also more basic, with a limited set of repeat modules mixed and matched to generate the startling structural and dynamic complexity observed.
Materials and methods
Sequence analysis
Secondary structure prediction of human Nup133 was performed with the PredictProtein server (http://cubic.bioc.columbia.edu/predictprotein), disordered region prediction with Pondr (http://www.pondr.com), and multiple alignment of homologous Nup133 sequences with ClustalW. Regions of secondary structure and homology separated by disordered, nonhomologous regions of >40 amino acids were considered separate domains.
In vitro binding assays
Full-length and mutant Nup133 proteins (amino acids 11156, 1514, and 4971156) were in vitro transcribed and translated in the presence of [35S]methionine by a coupled reticulocyte lysate transcription/translation system (TNT T7; Promega). Binding assays were performed essentially as described previously (Yaseen and Blobel, 1999) using recombinant full-length Nup107 fused to GST and in vitro transcribed/translated Nup133 proteins as indicated in the figure legends. Bound and unbound fractions were resolved by SDS-PAGE and analyzed by autoradiography.
Transfection and immunofluorescence microscopy
HeLa cells were grown in DME (Invitrogen) supplemented with 10% FBS, penicillin, and streptomycin. For transfections and immunofluorescence microscopy, cells were grown on coverslips and transfected using Effectene (QIAGEN) following the manufacturer's instructions. 24 h later, cells were washed twice with PBS and fixed/permeabilized in 100% methanol at 20°C for 5 min. Costaining with mAb414 (BAbCO) was performed as described previously (Boehmer et al., 2003) except that CY5-conjugated donkey mouse IgG antibodies (Jackson ImmunoResearch Laboratories) were used as secondary antibodies. Samples were examined using a spectral confocal microscope (model TCS SP; Leica). Images were processed in Adobe Photoshop CS.
Protein preparation and crystallization
Residues 67514 of human Nup133 with a His6 tag and thrombin cleavage site at the NH2 terminus were expressed in E. coli strain BL21 CodonPlus(DE3)-RIL cells at 23°C. Nup133 NTD was purified using a nickel affinity resin followed by thrombin cleavage overnight. The protein was further purified on a HiTrap Q anionic exchange column and gel filtration on a Superdex 75 column. Before crystallization, hNup133 NTD was concentrated to 10 mg ml1 in 20 mM Tris-Cl, pH 8.0, 150 mM NaCl, and 3 mM dithiothreitol. The protein was crystallized at 4°C with the hanging drop vapor diffusion method by mixing 2.5 µl of protein with 2.5 µl of precipitant solution, containing 1719% (wt/vol) PEG 3350, 100 mM Bis-Tris, pH 5.66.2, and 200 mM Li2SO4. Crystals were cryo protected in paraffin oil and frozen in liquid N2. Selenomethionine-substituted derivatives were prepared according to published protocols (Doublie, 1997).
Data collection, structure determination, and refinement
A SAD data set (to 2.5 Å) of an SeMet derivative was collected at 100 K at the 8.2.1 beamline of the Advanced Light Source. Two selenium sites (out of five possible) were identified with the program Shake-and-Bake (Weeks and Miller, 1999) and an additional two sites were found using the program SHARP (de la Fortelle and Bricogne, 1997). The resulting solvent-flattened electron density map was used to build an initial model that was refined using CNS (Brunger et al., 1998) and REFMAC5. The resulting model was refined against native data collected to 2.35 Å. The quality of the final model was validated using the program PROCHECK of the CCP4 suite (Collaborative Computational Project Number, 1994) and contains residues 75162, 170201, 206251, and 270477 and 97 water molecules. Coordinates and native structure factors were submitted to the Protein Data Bank with accession code 1XKS. Final statistics are provided in Table S1 (available at http://www.jcb.org/cgi/content/full/jcb.200408109/DC1).
Online supplemental material
Data and refinement statistics for the determination of hNup133 NTD are provided in Table S1. Video 1 shows the hNup133 ß-propeller rotating top to bottom, and then around its circumference. A multiple sequence alignment of Nup133 NTDs across representative vertebrates, insects, worms, and fungi is provided in Fig. S1. Online supplemental material is available at http://www.jcb.org/cgi/content/full/jcb.200408109/DC1.
Acknowledgments
We thank J. Glavy for advice and discussion, members of the Blobel laboratory for assistance and knowledge shared during this project, M. Rout for stimulating discussions and comments, and the staff at Advanced Light Source beamline 8.2.1 for technical assistance.
Submitted: 18 August 2004
Accepted: 15 October 2004
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