Structure, dynamics, evolution, and function of a major scaffold component in the nuclear pore complex - PubMed (original) (raw)

. 2013 Apr 2;21(4):560-71.

doi: 10.1016/j.str.2013.02.005. Epub 2013 Mar 14.

Seung Joong Kim, Paula Upla, William J Rice, Jeremy Phillips, Benjamin L Timney, Ursula Pieper, Jeffrey B Bonanno, Javier Fernandez-Martinez, Zhanna Hakhverdyan, Natalia E Ketaren, Tsutomu Matsui, Thomas M Weiss, David L Stokes, J Michael Sauder, Stephen K Burley, Andrej Sali, Michael P Rout, Steven C Almo

Affiliations

Structure, dynamics, evolution, and function of a major scaffold component in the nuclear pore complex

Parthasarathy Sampathkumar et al. Structure. 2013.

Abstract

The nuclear pore complex, composed of proteins termed nucleoporins (Nups), is responsible for nucleocytoplasmic transport in eukaryotes. Nuclear pore complexes (NPCs) form an annular structure composed of the nuclear ring, cytoplasmic ring, a membrane ring, and two inner rings. Nup192 is a major component of the NPC's inner ring. We report the crystal structure of Saccharomyces cerevisiae Nup192 residues 2-960 [ScNup192(2-960)], which adopts an α-helical fold with three domains (i.e., D1, D2, and D3). Small angle X-ray scattering and electron microscopy (EM) studies reveal that ScNup192(2-960) could undergo long-range transition between "open" and "closed" conformations. We obtained a structural model of full-length ScNup192 based on EM, the structure of ScNup192(2-960), and homology modeling. Evolutionary analyses using the ScNup192(2-960) structure suggest that NPCs and vesicle-coating complexes are descended from a common membrane-coating ancestral complex. We show that suppression of Nup192 expression leads to compromised nuclear transport and hypothesize a role for Nup192 in modulating the permeability of the NPC central channel.

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Figures

Figure 1

Figure 1. Structure and Surface Properties of ScNup192(2–960)

(A) Overall structure of ScNup192(2–960) is shown as a cartoon representation with the D1, D2, and D3 domains colored cyan, green, and gold, respectively. Interdomain loops comprising residues 204–211 and 663–670 are marked by arrows. Residues at the boundaries of the disordered loops are depicted as gray spheres, and the beginning of these loops are marked as L1, L2, L3, and L4. Side panels show individual D1, D2, and D3 domains as the blue to red rainbow from N to C terminus. Secondary structure elements are shown as defined by the DSSP (Kabsch and Sander, 1983) program. Strands of the β-hairpin are labeled in blue; additionally, helices are numbered consecutively with 310 helices treated as α helices for simplicity. See also Figure S1. (B) Representations of the front, rear, convex, and concave views of the ScNup192(2–960) are illustrated with the D1, D2, and D3 domains colored cyan, green, and gold, respectively. (C) The top row shows electrostatic potential of ScNup192(2–960) plotted onto its solvent accessible surface. Missing side chains and charges were assigned for ScNup192(2–960) structure using Protein Data Bank (PDB) 2PQR (Dolinsky et al., 2007), and electrostatic surface was calculated using APBS (Baker et al., 2001) within PyMOL. Negative (−7 kT/e) and positive (+7 kT/e) potentials are shown in red and blue, respectively. The bottom row shows conservation of 11 different fungal Nup192 sequences plotted onto the surface of ScNup192(2–960) structure with least to absolutely conserved residues colored as a gradient from white to orange. See also Figure S2.

Figure 2

Figure 2. Conformational Flexibility of ScNup192(2–960)

(A) Stereoview of the top normal mode predicted by elNémo (Suhre and Sanejouand, 2004) is shown, depicting the motion of D1 and D3 domains relative to the D2 domain of ScNup192(2–960). Residues in the hinge regions 204–211 and 663–670, between the domains, are colored in black. (B) Comparison of the merged experimental SAXS profile (black) of ScNup192(2–960) with the calculated SAXS profiles from the crystal structure (χ = 4.03, red), the “complete model” (χ = 3.84, green), and the 3:2 mixture of “open” and “closed” conformations (χ = 1.45, blue) are shown. The lower plot presents the residuals (calculated intensity/experimental intensity) of each calculated SAXS profile. The upper inset shows the SAXS profiles in the Guinier plot with an Rg fit of 39.30 ± 0.45 Å. The maximum particle size (_D_max) is 123 Å. (C) The crystal structure shown as cylinders (in red, front and top views) was fitted to the ab initio shape (represented as a gray envelope) computed from the experimental SAXS profile. This comparison reveals extra volume in solution. See also Figure S3A. (D) The “open” (in blue) and “closed” (in cyan) conformations of ScNup192(2–960) were fitted with the ab initio shape (represented as a gray envelope) computed from the experimental SAXS profile. The mixture of two conformation envelopes agrees well with the ab initio shape. See also Figure S3B and Movies S1, S2, S3, and S4. (E) Selected negative stain EM class averages are shown along with the projections of the “complete model”, “open”, and “closed” conformations. The em2D scores are shown in the bottom of each projection. See also Figure S4, showing 44 EM class averages along with projection of the “complete model”, “open”, and “closed” conformations, and Figure S5, illustrating histogram of em2D scores for selected class averages. See also Tables S1 and S2.

Figure 3

Figure 3. Structural Relatives of ScNup192(2–960)

(A) The heat map shown above illustrates the structural relationship of ScNup192(2–960) with selected α-helical proteins representing ten functional groups. Standardized scores for Dali, CE, and Multiprot alignments represented as a yellow to red gradient; red indicates stronger alignment scores (Table S2). The structure dendrogram is computed by hierarchical clustering using pairwise distances between alignment Z scores. The bar on the left shows the protein class for each structure, showing that karyopherins, β-catenin, and adaptin proteins are closest to Nup192. See also Figures S6B and S6C and Tables S4 and S5 for analyses on Nup85 and Nup170 structures. (B–E) Structural superpositions of Nup192(2–960) with karyopherin 60 (PDB: 1EE4, chain A), karyopherin 95 (PDB: 1IBR, chain B), β-catenin (PDB: 1QZ7, chain A), and adaptor protein 1 (PDB: 1W63, chain A), respectively, are illustrated based on DALI alignments. These were the top structural alignment hits among the karyopherin alpha, karyopherin beta, β-catenin, and adaptin protein families. The D1, D2, and D3 domains of ScNup192(2–960) structure are shown in cyan, green, and gold, respectively, and the superposed molecules are in gray. See also Figure S6A. See also Table S3.

Figure 4

Figure 4. Structural Model of the ScNup192FL

The EM reconstruction of ScNup192FL refined at 26 Å resolution, represented as envelope, is shown in two distinct views. The crystal structure of ScNup192(2–960) and a homology model of ScNup192 C-terminal region, residues 961–1,683, were fitted on the EM map using Chimera (Pettersen et al., 2004). The D1, D2, and D3 domains of ScNup192(2–960) are shown in cyan, green, and gold, respectively. The homology model of ScNup192 C-terminal region is shown in gray. See also Figure S7 and Table S6.

Figure 5

Figure 5. Steady State Localization of Transport Reporter after Depletion of Nup192

(A) The different panels show the localization of the Nab2NLS-mCherry-PrA NPC function reporter protein in strains where Nup192, Nup145, or Nup82 levels were repressed with chlortetracycline. Example images of these strains are shown after 0 or 24 hr of repression. (B) The N/C ratios of the reporter were quantified from ~100 cells per condition and normalized to a WT N/C value of 1 at the respective time point. These mean values are plotted with error bars of the standard error of the normalized means. Reported p values were the result of a Mann-Whitney rank sum test, comparing the distributions of normalized N/C values with or without repression. See also Figure S8.

Figure 6

Figure 6. Evolutionary Relationship between Membrane-Coating Complexes

Two possible evolutionary scenarios based on the structural relationship of ScNup192(2–960) with the karyopherin, β-catenin, and adaptin protein families are shown in (A) and (B). The α-solenoid-like and β-propeller folds are represented as rods and circles, respectively. See Discussion for the explanation.

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