Molecular Architecture of the Major Membrane Ring Component of the Nuclear Pore Complex - PubMed (original) (raw)

. 2017 Mar 7;25(3):434-445.

doi: 10.1016/j.str.2017.01.006. Epub 2017 Feb 2.

Seung Joong Kim 2, Parthasarathy Sampathkumar 3, Kaushik Dutta 4, Sean M Cahill 3, Ilan E Chemmama 2, Rosemary Williams 5, Jeffrey B Bonanno 3, William J Rice 6, David L Stokes 7, David Cowburn 3, Steven C Almo 3, Andrej Sali 8, Michael P Rout 9, Javier Fernandez-Martinez 10

Affiliations

Molecular Architecture of the Major Membrane Ring Component of the Nuclear Pore Complex

Paula Upla et al. Structure. 2017.

Abstract

The membrane ring that equatorially circumscribes the nuclear pore complex (NPC) in the perinuclear lumen of the nuclear envelope is composed largely of Pom152 in yeast and its ortholog Nup210 (or Gp210) in vertebrates. Here, we have used a combination of negative-stain electron microscopy, nuclear magnetic resonance, and small-angle X-ray scattering methods to determine an integrative structure of the ∼120 kDa luminal domain of Pom152. Our structural analysis reveals that the luminal domain is formed by a flexible string-of-pearls arrangement of nine repetitive cadherin-like Ig-like domains, indicating an evolutionary connection between NPCs and the cell adhesion machinery. The 16 copies of Pom152 known to be present in the yeast NPC are long enough to form the observed membrane ring, suggesting how interactions between Pom152 molecules help establish and maintain the NPC architecture.

Keywords: Gp210; NMR; Nup210; Pom152; SAXS; cadherin; electron microscopy; integrative structure determination; nuclear pore complex; nucleoporin.

Copyright © 2017 Elsevier Ltd. All rights reserved.

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Conflict of interest statement

Competing Financial Interests

The authors declare no competing financial interest.

Figures

Figure 1

Figure 1. Negative stain EM analysis shows that Pom152 has an extended, string-of-pearls shaped luminal domain

(A) Domain organization of Pom152FL and four truncations drawn to scale. Pom152FL exhibits a three-domain organization with the domain inside the NPC (NPC), the transmembrane segment (TM), and the domain inside the nuclear envelope lumen (luminal domain). The numbers indicate amino acid residue positions. Horizontal grey lines for the truncations represent the number of amino acid residues in each segment. (B) 35 representative negative-stain EM class averages of Pom152FL. The number of particles in each class is shown. Bar, 10 nm. (C) Representative random conical tilt 3D maps (right) are aligned to each of the corresponding class averages (left). The number of particles in each class is shown. Bars, 10 nm. (D) Negative-stain EM density map of Pom152FL. The nine globular domains in C-terminal lumen (1 to 9) and the N-terminal head region containing the NPC-associating and TM domains are indicated. Bar, 50 Å. (E) The average inter-domain angle for the last 7 repetitive regions was estimated in 37 representative Pom152FL class averages using the ImageJ angle tool. One angle was measured between domains 3 and 9, its difference from 180° was determined and the resulting value divided between the seven globular domains involved in the estimation. Distribution of the resulting inter-domain angles is shown in a Kernel density plot with a peak of −4.1° indicating a small negative curvature for the particles. (F) Representative negative-stain EM class averages of three assigned views of Pom152FL, Pom1521–1135, and Pom1521–936. The number of particles in each class is shown. Bar, 10 nm. (G) End-to-end distances of Pom152FL, Pom1521–1135, and Pom1521–936 in samples of 30, 28, and 33 class averages, respectively. The lines on the data points indicate the mean and SD: 38.7 ±1.5 nm, 28.9 ±1.6 nm and 24.4 ±1.0 nm for the samples, respectively. See also Figure S1 and Table S2.

Figure 2

Figure 2. Functional analysis of truncations affecting the Pom152 luminal domain

(A) Growth phenotypes of Pom152FL tagged with Protein A or prescission protease cleavage site (PPX)-Protein A and related truncation mutants (see Figure 1A). Serial 10-fold dilutions of cells were spotted on YPD plates in the absence or presence of 0.3% and 0.4% benzyl-OH at 30°C or on YPD plates an d grown at the indicated temperatures for 1–3 days. (B) Subcellular localization of benzyl-OH treated Pom152-GFP constructs. Panels show the localization of the indicated genomically tagged Pom152-GFP or Nup188-mCherry (used as NPC localization control) constructs as determined by fluorescence microscopy. Cells were grown on liquid yeast minimal media supplemented with 2% glucose at 30° C (untreated) or with an additional 0.1% benzyl-OH for 3 h at 30° C (benzyl-OH). Differential interference contrast (DIC). Bar, 5 microns. See also Figure 1A and Table S2.

Figure 3

Figure 3. NMR structure determination of Pom152718–820 reveals a conserved Ig-like fold domain

(A) Superimposition of 20 lowest-energy structures of Pom152718–820 that satisfy the NMR restraints best. The two β-sheets are made of the ABE (blue) and C′CFGG′A′ β-strands (cyan). (B) Topology of the Pom152718–820 fold. β-strands are represented as thick arrows and the linkers connecting them as lines. Each strand is named following the standard Ig-like domain annotation (Halaby et al., 1999). Scale bar, 5 Å. (C and D) Two views of the best-scoring structure, showing the arrangement of β-strands and features labeled as in panel B. (E and F) Sequence conservation of Pom152718–820 among 37 fungal homologs (Figure S6), plotted on the surface of the Pom152718–820 structure; low conservation in white, high conservation in red. (G and H) Electrostatic potential on the surface of the Pom152718–820 structure, calculated using the PyMOL tool APBS; negative (−2 kT/e) and positive (+2 kT/e) potentials are shown in red and blue, respectively. See also Figure S2 and Table 1.

Figure 4

Figure 4. Comparative models of 8 Ig-like domains and comparison to an Ig-like domain in human Nup210

(A) Domain organization of Pom152FL and its domains drawn to scale. The head region contains the domain that faces the NPC inner ring (NPC) and the transmembrane segment (TM), followed by the lumenal domain composed of nine Ig-like repeats. The amino acid boundaries for each Ig-like repeat are indicated below them. The repeat analyzed by NMR (Ig-4, Pom152718–820) is highlighted in light blue. (B) Comparative models of the remaining 8 Ig-like domains were built using the Pom152718–820 NMR structure as the template, followed by superposing them on the Pom152718–820 structure. The 8 domains share statistically significant sequence alignments to Pom152718–820 (E-values ranging from 3.3×10−49 to 6.8×10−39). (C) Sequence conservation among the 9 Ig-like domains was mapped on the structure of Pom152718–820 using ConSurf (Ashkenazy et al., 2016). Low conservation, white; high conservation, orange. (D) Domain organization of human Nup210. See legend of Figure 1A. (E) Superposition of comparative models for yeast Pom152718–820 (orange) and human Nup2101079–1152 (blue). See also Figure S3.

Figure 5

Figure 5. 4-stage scheme for integrative structure determination of Pom152FL

The integrative structure determination of Pom152FL proceeds through 4 stages: (1) gathering data, (2) representing and translating data into spatial restraints, (3) conformational sampling to produce an ensemble of structures that satisfies the restraints, and (4) analyzing, assessing, and validating the ensemble structures. The modeling protocol (i.e., stages 2, 3, and 4) was scripted using the Python Modeling Interface (PMI), version 4d97507, a library for modeling macromolecular complexes based on our open-source Integrative Modeling Platform (IMP) package, version 2.6 (

http://integrativemodeling.org

) (Russel et al., 2012).

Figure 6

Figure 6. Integrative structure of Pom152FL based on NMR spectroscopy, negative-stain EM, and SAXS

(A) (left) The localization probability density map computed from 364 superposed structures that satisfy the input spatial restraints shows the location of each of the 9 Ig-like domains (ranging from blue to red). The negative-stain EM density map is superposed in grey. A representative molecular model of Pom152LD (ribbon plot) was obtained by adjusting the relative orientations of adjacent Ig-like domains to resemble those in known cadherin structures (PDB codes 1L3W, 1EPF, 1NCI, 4ZI9, and 5K8R) (Boggon et al., 2002; Kasper et al., 2000; Nicoludis et al., 2015; Nicoludis et al., 2016; Shapiro et al., 1995). (right) Validation of the integrative structure of Pom152LD by SAXS data for five Pom152 segments, spanning residues 718–820 (Ig-4), 718–920 (Ig-4,5), 603–820 (Ig-3,4), 919–1020 (Ig-6), and 718–1148 (Ig-4,5,6,7). The shapes of these segments in our integrative structure match the envelopes (ab initio shapes) computed from the corresponding SAXS profiles. (B) Fit of 16 copies of Pom152LD into the yeast NPC map (Alber et al., 2007b). A good fit positions two copies of the extended Pom152LD molecule in an anti-parallel fashion on top of each other, forming a homodimer; we only show the potential arrangement of the antiparallel homodimer, which is implied by the C2-symmetry of the NPC (Alber et al., 2007b; Kosinski et al., 2016; Lin et al., 2016). See also Figures 5, S4–S7 and Table S1.

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