Architectural Nucleoporins Nup157/170 and Nup133 Are Structurally Related and Descend from a Second Ancestral Element
2009; Elsevier BV; Volume: 284; Issue: 41 Linguagem: Inglês
10.1074/jbc.m109.023580
ISSN1083-351X
AutoresJames R. Whittle, Thomas Schwartz,
Tópico(s)RNA modifications and cancer
ResumoThe nuclear pore complex (NPC) constitutes one of the largest protein assemblies in the eukaryotic cell and forms the exclusive gateway to the nucleus. The stable, ∼15–20-MDa scaffold ring of the NPC is built from two multiprotein complexes arranged around a central 8-fold axis. Here we present crystal structures of two large architectural units, yNup170979–1502 and hNup107658–925·hNup133517–1156, each a constituent of one of the two multiprotein complexes. Conservation of domain arrangement and of tertiary structure suggests that Nup157/170 and Nup133 derived from a common ancestor. Together with the previously established ancestral coatomer element (ACE1), these two elements constitute the major α-helical building blocks of the NPC scaffold and define its branched, lattice-like architecture, similar to vesicle coats like COPII. We hypothesize that the extant NPC evolved early during eukaryotic evolution from a rudimentary structure composed of several identical copies of a few ancestral elements, later diversified and specified by gene duplication. The nuclear pore complex (NPC) constitutes one of the largest protein assemblies in the eukaryotic cell and forms the exclusive gateway to the nucleus. The stable, ∼15–20-MDa scaffold ring of the NPC is built from two multiprotein complexes arranged around a central 8-fold axis. Here we present crystal structures of two large architectural units, yNup170979–1502 and hNup107658–925·hNup133517–1156, each a constituent of one of the two multiprotein complexes. Conservation of domain arrangement and of tertiary structure suggests that Nup157/170 and Nup133 derived from a common ancestor. Together with the previously established ancestral coatomer element (ACE1), these two elements constitute the major α-helical building blocks of the NPC scaffold and define its branched, lattice-like architecture, similar to vesicle coats like COPII. We hypothesize that the extant NPC evolved early during eukaryotic evolution from a rudimentary structure composed of several identical copies of a few ancestral elements, later diversified and specified by gene duplication. The membrane-enveloped nucleus is the hallmark of the eukaryotic cell. Physical separation of nucleoplasm and cytoplasm necessitates sites for molecular exchange (1Tran E.J. Wente S.R. Cell. 2006; 125: 1041-1053Abstract Full Text Full Text PDF PubMed Scopus (410) Google Scholar, 2Weis K. Cell. 2003; 112: 441-451Abstract Full Text Full Text PDF PubMed Scopus (575) Google Scholar, 3D'Angelo M.A. Hetzer M.W. Trends Cell Biol. 2008; 18: 456-466Abstract Full Text Full Text PDF PubMed Scopus (285) Google Scholar). Nuclear pore complexes (NPCs), 2The abbreviations used are: NPCnuclear pore complexPEGpolyethylene glycolACEancestral coatomer elementnupsnucleoporinsFGphenylalanine-glycine. 2The abbreviations used are: NPCnuclear pore complexPEGpolyethylene glycolACEancestral coatomer elementnupsnucleoporinsFGphenylalanine-glycine. plugged into circular openings where inner and outer nuclear membranes fuse, perforate the nuclear envelope and form the sole gateway. The NPC is, at ∼50 MDa, one of the largest protein assemblies in the quiescent cell. It is modular, comprises ∼30 different proteins, termed nucleoporins (nups), and forms an 8-fold symmetric ring embedded in the nuclear envelope (4Schwartz T.U. Curr. Opin Struct. Biol. 2005; 15: 221-226Crossref PubMed Scopus (137) Google Scholar). In accord with the symmetry of the complex, each nucleoporin is present in 8 × n copies/NPC. nuclear pore complex polyethylene glycol ancestral coatomer element nucleoporins phenylalanine-glycine. nuclear pore complex polyethylene glycol ancestral coatomer element nucleoporins phenylalanine-glycine. The architecture of the NPC is roughly conserved among eukaryotes, measuring ∼100 nm in the outer diameter, with a central transport gate ∼40 nm wide (5Panté N. Kann M. Mol. Biol. Cell. 2002; 13: 425-434Crossref PubMed Scopus (633) Google Scholar, 6Beck M. Lucić V. Förster F. Baumeister W. Medalia O. Nature. 2007; 449: 611-615Crossref PubMed Scopus (283) Google Scholar, 7Stoffler D. Feja B. Fahrenkrog B. Walz J. Typke D. Aebi U. J. Mol. Biol. 2003; 328: 119-130Crossref PubMed Scopus (192) Google Scholar, 8Beck M. Förster F. Ecke M. Plitzko J.M. Melchior F. Gerisch G. Baumeister W. Medalia O. Science. 2004; 306: 1387-1390Crossref PubMed Scopus (400) Google Scholar). The NPC is a highly dynamic assembly. Some nucleoporins are stably attached, whereas others are more dynamic (9Rabut G. Doye V. Ellenberg J. Nat. Cell Biol. 2004; 6: 1114-1121Crossref PubMed Scopus (355) Google Scholar, 10Rabut G. Lénárt P. Ellenberg J. Curr. Opin. Cell Biol. 2004; 16: 314-321Crossref PubMed Scopus (82) Google Scholar, 11Dultz E. Zanin E. Wurzenberger C. Braun M. Rabut G. Sironi L. Ellenberg J. J. Cell Biol. 2008; 180: 857-865Crossref PubMed Scopus (187) Google Scholar). The main scaffold ring is composed of ∼15 architectural nucleoporins that anchor to the inner pore wall. A second set of nucleoporins (FG-nups) is characterized by long, phenylalanine-glycine (FG) rich filamentous extensions. These FG fibers emanate into the central cavity of the NPC and define the main transport barrier (12Lim R.Y. Fahrenkrog B. Köser J. Schwarz-Herion K. Deng J. Aebi U. Science. 2007; 318: 640-643Crossref PubMed Scopus (235) Google Scholar, 13Frey S. Görlich D. 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The small GTPase Ran regulates the interaction of protein cargo with import or export karyopherins, conferring directionality to these transport processes. This regulation depends on Ran being GTP-bound in the nucleus and GDP-bound in the cytoplasm, a gradient established by the action of cytoplasmic GTPase-activating protein (RanGAP) and nuclear GTP exchange factor (RanGEF). To better understand the myriad of functions attributed to the NPC, which go far beyond transporting molecules across the nuclear envelope (18Fahrenkrog B. Köser J. Aebi U. Trends Biochem. Sci. 2004; 29: 175-182Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar, 19Lim R.Y. Ullman K.S. Fahrenkrog B. Int. Rev. Cell Mol. Biol. 2008; 267: 299-342Crossref PubMed Scopus (64) Google Scholar), we are interested in the structural characterization of the NPC, which begins with the stable scaffold structure. The ∼15 architectural nucleoporins are organized in two large multiprotein complexes: the well studied Nup84 complex and the more enigmatic Nic96 complex. The components of each are known (Table 1). The Nup84 complex contains seven universally conserved nucleoporins and adopts a characteristically branched Y shape (20Lutzmann M. Kunze R. Buerer A. Aebi U. Hurt E. EMBO J. 2002; 21: 387-397Crossref PubMed Scopus (178) Google Scholar, 21Siniossoglou S. Lutzmann M. Santos-Rosa H. Leonard K. Mueller S. Aebi U. Hurt E. J. Cell Biol. 2000; 149: 41-54Crossref PubMed Scopus (143) Google Scholar, 22Siniossoglou S. Santos-Rosa H. Rappsilber J. Mann M. Hurt E. EMBO J. 1998; 17: 6449-6464Crossref PubMed Scopus (165) Google Scholar). In this work, the Nup84 complex is referred to as the Y complex. Nup120 and Nup85·Seh1 form the two short arms, whereas Nup145C·Sec13, Nup84, and Nup133 build the long, kinked stalk. The Nic96 complex likely contains five distinct nucleoporins, two of them duplicated in yeast (23Grandi P. Dang T. Pané N. Shevchenko A. Mann M. Forbes D. Hurt E. Mol. Biol. Cell. 1997; 8: 2017-2038Crossref PubMed Scopus (126) Google Scholar, 24Miller B.R. Powers M. Park M. Fischer W. Forbes D.J. Mol. Biol. Cell. 2000; 11: 3381-3396Crossref PubMed Scopus (46) Google Scholar, 25Zabel U. Doye V. Tekotte H. Wepf R. Grandi P. Hurt E.C. J. Cell Biol. 1996; 133: 1141-1152Crossref PubMed Scopus (86) Google Scholar, 26Aitchison J.D. Rout M.P. Marelli M. Blobel G. Wozniak R.W. J. Cell Biol. 1995; 131: 1133-1148Crossref PubMed Scopus (162) Google Scholar, 27Marelli M. Aitchison J.D. Wozniak R.W. J. Cell Biol. 1998; 143: 1813-1830Crossref PubMed Scopus (133) Google Scholar). It connects to the nuclear envelope (28Weis K. Cell. 2007; 130: 405-407Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar) as well as the FG network (29Grandi P. Emig S. Weise C. Hucho F. Pohl T. Hurt E.C. J. Cell Biol. 1995; 130: 1263-1273Crossref PubMed Scopus (89) Google Scholar). These two scaffold complexes likely form ring-like assemblies. Whether these rings are stacked or concentric or arranged some other way is controversial (30Alber F. Dokudovskaya S. Veenhoff L.M. Zhang W. Kipper J. Devos D. Suprapto A. Karni-Schmidt O. Williams R. Chait B.T. Sali A. Rout M.P. Nature. 2007; 450: 695-701Crossref PubMed Scopus (818) Google Scholar, 31Hsia K.C. Stavropoulos P. Blobel G. Hoelz A. Cell. 2007; 131: 1313-1326Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar, 32Brohawn S.G. Leksa N.C. Spear E.D. Rajashankar K.R. Schwartz T.U. Science. 2008; 322: 1369-1373Crossref PubMed Scopus (169) Google Scholar, 33Brohawn S.G. Schwartz T.U. Commun. Integr. Biol. 2009; 2: 1-3Crossref PubMed Scopus (16) Google Scholar). This structural framework is important to the assembly and function of the NPC. Severe defects occur when scaffold nucleoporins are deleted or depleted, including failure to recruit other nucleoporins and diminished transport of protein or RNA across the nuclear membrane (34Doye V. Wepf R. Hurt E.C. EMBO J. 1994; 13: 6062-6075Crossref PubMed Scopus (207) Google Scholar, 35Heath C.V. Copeland C.S. Amberg D.C. Del Priore V. Snyder M. Cole C.N. J. Cell Biol. 1995; 131: 1677-1697Crossref PubMed Scopus (112) Google Scholar, 36Li O. Heath C.V. Amberg D.C. Dockendorff T.C. Copeland C.S. Snyder M. Cole C.N. Mol. Biol. Cell. 1995; 6: 401-417Crossref PubMed Scopus (79) Google Scholar, 37Vasu S. Shah S. Orjalo A. Park M. Fischer W.H. Forbes D.J. J. Cell Biol. 2001; 155: 339-354Crossref PubMed Scopus (132) Google Scholar, 38Harel A. Orjalo A.V. Vincent T. Lachish-Zalait A. Vasu S. Shah S. Zimmerman E. Elbaum M. Forbes D.J. Mol. Cell. 2003; 11: 853-864Abstract Full Text Full Text PDF PubMed Scopus (221) Google Scholar, 39Gao H. Sumanaweera N. Bailer S.M. Stochaj U. J. Biol. 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PLoS Biol. 2004; 2: e380Crossref PubMed Scopus (318) Google Scholar). Experimental structural characterization, however, has revealed that this simplistic description does not adequately reflect the reality. For example, Sec13 and Seh1 are predicted as six-bladed β-propellers but turn out to be seven-bladed, with the final blade provided in trans by their respective binding partners (31Hsia K.C. Stavropoulos P. Blobel G. Hoelz A. Cell. 2007; 131: 1313-1326Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar, 32Brohawn S.G. Leksa N.C. Spear E.D. Rajashankar K.R. Schwartz T.U. Science. 2008; 322: 1369-1373Crossref PubMed Scopus (169) Google Scholar, 44Fath S. Mancias J.D. Bi X. Goldberg J. Cell. 2007; 129: 1325-1336Abstract Full Text Full Text PDF PubMed Scopus (227) Google Scholar, 45Debler E.W. Ma Y. Seo H.S. Hsia K.C. Noriega T.R. Blobel G. Hoelz A. Mol. Cell. 2008; 32: 815-826Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). The four ACE1 nucleoporins are built around a ∼65-kDa α-helical domain. They are distantly related to one another and, strikingly, also to Sec31, the main structural component of the outer coat of the COPII vesicle. This ACE1 domain is a tripartite fold back structure of ∼28 α-helices, distinct from the regular α-solenoid domains found in HEAT, TPR, or PPR repeat proteins (46Andrade M.A. Perez-Iratxeta C. Ponting C.P. J. Struct. Biol. 2001; 134: 117-131Crossref PubMed Scopus (470) Google Scholar, 47Andrade M.A. Petosa C. O'Donoghue S.I. Müller C.W. Bork P. J. Mol. Biol. 2001; 309: 1-18Crossref PubMed Scopus (396) Google Scholar), among others. The structural similarity between these ACE1 proteins provided the proof that the NPC and COPII coat derive from a common ancestor (32Brohawn S.G. Leksa N.C. Spear E.D. Rajashankar K.R. Schwartz T.U. Science. 2008; 322: 1369-1373Crossref PubMed Scopus (169) Google Scholar), as hypothesized previously (42Devos D. Dokudovskaya S. Williams R. Alber F. Eswar N. Chait B.T. Rout M.P. Sali A. Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 2172-2177Crossref PubMed Scopus (225) Google Scholar, 43Devos D. Dokudovskaya S. Alber F. Williams R. Chait B.T. Sali A. Rout M.P. PLoS Biol. 2004; 2: e380Crossref PubMed Scopus (318) Google Scholar). The ACE1 nucleoporin Nup84 binds Nup133. The structure of a fragment of the human Nup84 ortholog, hNup107658–925, has been solved in complex with hNup133934–1156 (48Boehmer T. Jeudy S. Berke I.C. Schwartz T.U. Mol. Cell. 2008; 30: 721-731Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar), the C terminus of the protein. This structure showed that the C terminus of Nup133 consists of two α-helical blocks. A rigid block of four α-helices, residues 934–1008, forms an interface bundle that binds Nup84(hNup107). A moderately flexible hinge connects this interface bundle to a second α-helical unit that forms a distinct lobe at the C terminus of the protein. The N terminus of Nup133 is a β-propeller, whose structure is also known (49Berke I.C. Boehmer T. Blobel G. Schwartz T.U. J. Cell Biol. 2004; 167: 591-597Crossref PubMed Scopus (94) Google Scholar). hNup133934–1156 suggested that Nup133 is not an ACE1 protein. Here we present crystallographic analysis of two architectural units, yNup170979–1502 and hNup107658–925·hNup133517–1156, components of both major scaffold complexes of the NPC. Nup170, its paralog Nup157, and Nup133 each consist of an N-terminal β-propeller followed by an ∼80-kDa C-terminal α-helical domain. The structures reveal a common α-helical architecture for Nup157/170 and Nup133 that is distinct from all other known nucleoporins. This α-helical architecture is, with ACE1, another ancestral element of the NPC. We conclude that the basic NPC framework is built from a small set of recognizable structural elements that were already present in multiple copies in the last common ancestor of extant eukaryotes. During the course of evolution, gene duplications occurred and diversified these core elements, generating the complex, multi-functional machine that is the NPC. Nup170 from Saccharomyces cerevisiae was cloned into a pET-Duet vector (Novagen) encoding an N-terminal, human rhinovirus 3C (HR3C)-cleavable His6 tag. N-terminal truncations were generated by PCR methods. Nup133 (residues 517–1156) and Nup107 (residues 658–925) from Homo sapiens were cloned into a bicistronic pET-Duet vector modified to encode N-terminal, thrombin-cleavable His6 tags. The proteins were expressed in Escherichia coli strain BL21 (DE3)-RIL (Stratagene) in LB medium, induced with 200 μm isopropyl β-d-1-thiogalactopyranoside at 18 °C. Cells expressing yNup170979–1502 or yNup1701253–1502 were homogenized at 4 °C in 50 mm potassium phosphate, pH 8.5, 400 mm NaCl, 40 mm imidazole, 5 mm β-mercaptoethanol. The protein was bound to nickel affinity resin; eluted with 250 mm imidazole; dialyzed against 20 mm Tris-HCl, pH 8.5, 150 mm NaCl, 0.5 mm EDTA, 1 mm dithiothreitol; and purified, after affinity tags were removed, on a Superdex S75 column (GE Healthcare), equilibrated in 10 mm Tris-HCl, pH 8.5, 150 mm NaCl, 0.1 mm EDTA, 1 mm dithiothreitol. Selenomethionine-substituted protein was expressed as described (32Brohawn S.G. Leksa N.C. Spear E.D. Rajashankar K.R. Schwartz T.U. Science. 2008; 322: 1369-1373Crossref PubMed Scopus (169) Google Scholar). hNup107658–925·hNup133517–1156 were bound to nickel affinity resin in lysis buffer 20 mm Tris-HCl, 5 mm potassium phosphate, pH 8.5, 250 mm NaCl, 10 mm imidazole, 5 mm β-mercaptoethanol; then eluted with 250 mm imidazole; and purified, after the affinity tags were removed, on a HiTrap FF and then a Superdex 200 column equilibrated in 5 mm potassium phosphate, pH 7, 150 mm NaCl, 0.1 mm EDTA, 1 mm dithiothreitol. yNup170979–1502 was concentrated to 4–8 mg ml−1. An initial crystallization condition was found by vapor diffusion using commercial screens. Crystallization was greatly improved by the addition of tris(2-carboxyethyl)phosphine. Crystals were grown at 4 °C, in 2-μl hanging drops over 0.2 m ammonium acetate, 0.1 m Tris-HCl, pH 7.9, 5–10% polyethylene glycol (PEG) 3,350, 5 mm tris(2-carboxyethyl)phosphine. Rods, ∼80–100 × ∼80–100 × 300–400 μm, with isosceles triangular bases, formed within 3 days. They were flash-frozen in reservoir supplemented with 24% PEG 200, 24% ethylene glycol, or 25% glycerol. yNup1701253–1502 crystallized at 90 mg ml−1 in 1-μl hanging drops over 0.1 m Tris-HCl, pH 8.5, 0.2 m Li2SO4, 50 mm NaCl, 22–24% PEG 3,350 within 3–5 days at 18 °C. Plates, 50 × 300 × 300 μm, were cryoprotected in reservoir solution supplemented with 12% glycerol and flash-frozen. hNup107658–925·hNup133517–1156 was concentrated to 9 mg ml−1, and 2% PEG 3,350 was added. An initial crystallization condition was found using commercial screens. After optimization, the crystals were grown in drops of 1 μl of protein, supplemented with 2% PEG 3,3350, + 1 μl of reservoir of 0.8–1.0 m sodium/potassium phosphate, pH 7.8, 15% glycerol at 18 °C, streak-seeded after 12 h with microcrystals. In most drops, phase separation rather than crystallization was observed. Occasionally, thin needles with dimensions of 30 × 30 × 150 μm grew within 2 days. The crystals were retrieved on MicroMounts (Mitegen) and flash-frozen in liquid nitrogen. The data for yNup170979–1502 were collected at 100 K at microfocus Beamline 24-IDE at the Advanced Photon Source (Argonne, IL). The crystals that diffracted best, to ∼2.5 Å, were perfectly merohedrally twinned. Untwinned data were obtained to 3.2 Å and used for further analysis. The data were collected from selenomethionine-labeled crystals and processed with the HKL2000 package (50Otwinowski Z. Minor W. Macromol. Crystallogr. A. 1997; 276: 307-326Crossref Scopus (38526) Google Scholar). The phases were determined by selenium single-wavelength anomalous dispersion. Nine of ten possible selenium sites were identified with SHELXD (51Sheldrick G.M. Acta Crystallogr. A. 2008; 64: 112-122Crossref PubMed Scopus (80558) Google Scholar). Selenium positions were refined with SHARP (52Vonrhein C. Blanc E. Roversi P. Bricogne G. Methods Mol. Biol. 2007; 364: 215-230PubMed Google Scholar), which also revealed the additional selenium site, and experimental phases were calculated. The resulting solvent-flattened electron density map was used to build a model with Coot (53Emsley P. Cowtan K. Acta Crystallogr. D Biol. Crystallogr. 2004; 60: 2126-2132Crossref PubMed Scopus (23226) Google Scholar). To improve model quality and attempt to refine against the twinned data diffracting to higher resolution, the C-terminal subdomain yNup1701253–1502 was crystallized, and 2.2 Å data was collected at Beamline 24-IDC at the Advanced Photon Source from a large crystal formed from several caked layers. The strongest of the observed diffraction patterns was indexed and integrated. Of many specimens tested, morphologically indistinguishable, only this crystal diffracted strongly and belonged to space group I222 (Table 2). All others belonged to space group P6322, diffracted to 3.5 Å, and were not further analyzed. A molecular replacement solution for yNup1701253–1502 was found using a partial model from the initially obtained 3.2 Å structure of yNup170979–1502. A complete model for yNup1701253–1502 was built automatically, with minor intervention, using PHENIX (54Adams P.D. Grosse-Kunstleve R.W. Hung L.W. Ioerger T.R. McCoy A.J. Moriarty N.W. Read R.J. Sacchettini J.C. Sauter N.K. Terwilliger T.C. Acta Crystallogr. D Biol. Crystallogr. 2002; 58: 1948-1954Crossref PubMed Scopus (3624) Google Scholar). yNup170979–1502 was rebuilt incorporating this partial model, improving refinement parameters. We note, however, that the model of the yNup170 C-terminal subdomain did not help process the twinned 2.5 Å data (not shown).TABLE 2Data collection and refinement statistics Data for hNup107658–925·hNup133517–1156 were collected at 100 K at 24-IDE. Because of significant radiation damage, partial data sets were collected and merged from several crystals grown in the same crystallization drop, each exposed at 3–10 spots. The structure was phased by molecular replacement using the minimal 55-kDa hNup107658–925·hNup133934–1156 interaction complex (48Boehmer T. Jeudy S. Berke I.C. Schwartz T.U. Mol. Cell. 2008; 30: 721-731Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar) as a search model in Phaser (55McCoy A.J. Grosse-Kunstleve R.W. Adams P.D. Winn M.D. Storoni L.C. Read R.J. J. Appl. Crystallogr. 2007; 40: 658-674Crossref PubMed Scopus (14440) Google Scholar) in the CCP4 suite (56Bailey S. Acta Crystallogr. D Biol. Crystallogr. 1994; 50: 760-763Crossref PubMed Scopus (41) Google Scholar). The additional 45-kDa domain was built and refined with Coot (53Emsley P. Cowtan K. Acta Crystallogr. D Biol. Crystallogr. 2004; 60: 2126-2132Crossref PubMed Scopus (23226) Google Scholar) and PHENIX (54Adams P.D. Grosse-Kunstleve R.W. Hung L.W. Ioerger T.R. McCoy A.J. Moriarty N.W. Read R.J. Sacchettini J.C. Sauter N.K. Terwilliger T.C. Acta Crystallogr. D Biol. Crystallogr. 2002; 58: 1948-1954Crossref PubMed Scopus (3624) Google Scholar). The data to 3.5 Å were included, despite low I/ςI, as recommended for low resolution crystallography (57Brunger A.T. DeLaBarre B. Davies J.M. Weis W.I. Acta Crystallogr. D Biol. Crystallogr. 2009; 65: 128-133Crossref PubMed Scopus (47) Google Scholar). Anisotropic diffraction was corrected by elliptical resolution truncation and anisotropic B-factor correction using the Diffraction Anisotropy Server (58Strong M. Sawaya M.R. Wang S. Phillips M. Cascio D. Eisenberg D. Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 8060-8065Crossref PubMed Scopus (574) Google Scholar). The obtained electron density maps allowed positioning of the secondary structure elements, which are essentially all α-helical. Connections between helices were mostly visible, allowing tracing of the molecule from the N to the C termini. Observed chain topology and variation in the length of helices allowed us to assign each modeled helix unambiguously to the secondary structure as predicted by the PredictProtein server (59Rost B. Yachdav G. Liu J. Nucleic Acids Res. 2004; 32: 321-326Crossref PubMed Scopus (1180) Google Scholar). In the absence of detailed positional markers, the assigned sequence in the deposited data is approximate but is likely erroneous only in a few places and shifted by not more than three or four residues, i.e. one α-helical turn. Several nonhelical loops could be traced confidently, including loops that are disordered in the partial structure hNup107658–925·hNup133934–1156 (48Boehmer T. Jeudy S. Berke I.C. Schwartz T.U. Mol. Cell. 2008; 30: 721-731Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). Nup170 homologs were retrieved from the NCBI website data base, and a multiple sequence alignment was calculated by the MUSCLE algorithm (60Edgar R.C. Nucleic Acids Res. 2004; 32: 1792-1797Crossref PubMed Scopus (30224) Google Scholar). An average distance tree was used to select representative, divergent sequences. The residues were scored for conservation by the AMAS method in JALVIEW (61Waterhouse A.M. Procter J.B. Martin D.M. Clamp M. Barton G.J. Bioinformatics. 2009; 25: 1189-1191Crossref PubMed Scopus (5690) Google Scholar). PBD2PQR (62Dolinsky T.J. Nielsen J.E. McCammon J.A. Baker N.A. Nucleic Acids Res. 2004; 32: 665-667Crossref PubMed Scopus (2510) Google Scholar) and APBS (63Baker N.A. Sept D. Joseph S. Holst M.J. McCammon J.A. Proc. Natl. Acad. Sci. U.S.A. 2001; 98: 10037-10041Crossref PubMed Scopus (5845) Google Scholar) software packages were used to calculate surface charge, and the PISA server was used to calculate the accessible surface area (64Krissinel E. Henrick K. J. Mol. Biol. 2007; 372: 774-797Crossref PubMed Scopus (6769) Google Scholar). MODELLER (65Eswar N. Webb B. Marti-Renom M.A. Madhusudhan M.S. Eramian D. Shen M.Y. Pieper U. Sali A. Curr. Protoc. Protein Sci., Unit 2.9. 2007; 50: 2.9.1-2.9.31Crossref Google Scholar) was used to build a complete model of yNup157900–1391. Pymol was used to generate figures. Nup170 is predicted to contain two structural domains: an N-terminal β-propeller (residues 180–650) and a C-terminal α-helical domain (Fig. 1A) (4Schwartz T.U. Curr. Opin Struct. Biol. 2005; 15: 221-226Crossref PubMed Scopus (137) Google Scholar, 42Devos D. Dokudovskaya S. Williams R. Alber F. Eswar N. Chait B.T. Rout M.P. Sali A. Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 2172-2177Crossref PubMed Scopus (225) Google Scholar). By expressing a series of N-terminal truncations of the protein and by limited proteolysis, we defined a stable core of the predicted α-helical region, comprising residues 979–1502 of Nup170 from S. cerevisiae (data not shown). The presumed N-terminal β-propeller domain interacts weakly with the α-helical domain when separated, indicating flexible attachment (66Flemming D. Sarges P. Stelter P. Hellwig A. Böttcher B. Hurt E. J. Cell Biol. 2009; 185: 387-395Crossref PubMed Scopus (29) Google Scholar). yNup170979–1502 was expressed recombinantly in E. coli, purified to homogeneity, and crystallized. The structure was solved by single-wavelength anomalous dispersion, using selenomethionine-labeled protein. The asymmetric unit contains one molecule. The experimental single-wavelength anomalous dispersion electron density allowed for building residues 1020–1460, revealing a continuous but bipartite stacked α-helical domain (Fig. 1). Because of the lack of strong crystal contacts, the C-terminal half of the domain is flexibly positioned. Thus, to aid structure determination, this C-terminal 29-kDa subdomain (residues 1253–1502) was separately expressed and crystallized. The data to 2.2-Å resolution were collected and phased by molecular replacement with the relevant portion of the larger protein as initially modeled. The complete, refined model of the C-terminal subdomain (Rfree/Rwork = 27.6/23.3%) was then used to build the structure of yNup170979–1502 at 3.2-Å resolution (Rfree/Rwork = 32.4/30.6%). The representative electron density for the 3.2-Å resolution structure is shown in supplemental Fig. S1. The crystal packing of the 2.2-Å resolution structure is shown in supplemental Movie S1. The data collection and refinement statistics are summarized in Table 2. yNup170979–1502 adopts an irregular α-helical stack composed of 26 α-helices and overall dimensions of 12 × 4 × 4 nm (Fig. 1). We label these helices α1–26. The domain begins with helices α1/2, α3/4, and α6/7 forming three consecutive pairs of helices of various lengths, stacked antiparallel, without superhelical twist. Helix α5 resides in a loop and does not pair to other helices. Helices α8–13 form an extended zigzag pattern that is rotated by ∼90° against the α1–7 stack. This zigzag can be likened to a stack of three α-helical pairs that has been stretched by pulling on its ends. As a result, helices α8–13 extend over ∼38 Å, reflecting a ∼50% stretch compared with a tightly packed six-helix stack, which would span only ∼26 Å. The hydrophobic core of this extended zigzag is poorly packed. Few residues are fully buried. Helix α14 is approximately twice as long as its direct neighbors and connects the two α-helical subdomains. The C-terminal subdomain forms a crescent only loosely definable as a stack. It starts with α15, unexpectedly positioned below, not above, helix α14. This helix abuts end-on-end to α12 of the N-terminal subdomain. The strictly conserved Arg1232 is sandwiched between the negatively polarized C termini of α12 and α15, presumably for charge compensation. Nup170 then continues with helices α16–26 forming a compact hydrophobic core, implying rigidity. To compare the structure of Nup170 with those of other proteins, we performed structure-based searches with VAST and DALI (67Madej T. Gibrat J.F. Bryant S.H. Proteins. 1995; 23: 356-369Crossref PubMed Scopus (373) Google Scholar, 68Holm L. Kääriäinen S. Rosenström P. Schenkel A. Bioinfo
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