Three-dimensional structure and flexibility of a membrane-coating module of the nuclear pore complex

Abstract

The nuclear pore complex mediates nucleocytoplasmic transport in all eukaryotes and is among the largest cellular assemblies of proteins, collectively known as nucleoporins. Nucleoporins are organized into distinct subcomplexes. We optimized the isolation of a putative membrane-coating subcomplex of the nuclear pore complex, the heptameric Nup84 complex, and analyzed its structure by EM. Our data confirmed the previously reported 'Y' shape. We discerned additional structural details, including specific hinge regions at which the particle shows great flexibility. We determined the three-dimensional structures of two conformers, mapped the localization of two nucleoporins within the subcomplex and docked known crystal structures into the EM maps. The free ends of the Y-shaped particle are formed by beta-propellers; the connecting segments consist of alpha-solenoids. Notably, the same organizational principle is found in the clathrin triskelion, which may share a common evolutionary origin with the heptameric complex.

Figure 1

Purification and electron microscopy (EM) of the heptameric Nup84 complex. (a) Size exclusion chromatography profile of affinity-purified Nup84 complex released from IgG-beads by TEV protease cleavage. The fraction indicated by dashed lines contains Nup84 complex and was used for EM. (b) Coomassie-stained SDS-PAGE of the fraction from size exclusion chromatography indicated in (a). All bands were identified by mass spectrometry. Nup85-CBP is Nup85 C-terminally tagged with the calmodulin-binding peptide moiety of the TAP-tag, which was cleaved from the protein A moiety by TEV protease. (c) Negative-stain EM of heptameric complex particles. A field of particles adsorbed to glow-discharged carbon film and stained with 2% uranyl formate is shown. Scale bar, 100 nm.

Figure 2

Alignment, classification and averaging of particle images reveals structural details of the heptameric complex. (a) Reference-free alignment and k-means classification of 9,028 particle images into 90 classes resulted in the depicted class averages. The number of particles constituting each class is indicated. (b) A well-defined class average is shown and prominent features are named. Scale bar, 100 Å.

Figure 3

Heterogeneity of particle appearance. (a) Definition of angles between particle segments for the two-dimensional view (2D) of particles. α, angle between long and short arms; β, angle between long arm and vertex-proximal stem segment; γ, angle at stem hinge 1; δ, angle at stem hinge 2. (b) Distribution of angles for the 90 classes shown in Figure 2. Angles were measured for 2D class averages and assigned to the number of particles constituting each class. (c) Correlation between γ and δ. Each marker indicates γ and δ for a particle class. Marker area is proportional to class size. Particle-based correlation coefficient: 0.78 (shown in purple); Class-based correlation coefficient: 0.72. All other combinations of angles α, β, γ and δ are plotted in Supplementary Figure 1; the corresponding correlation coefficients are between −0.44 and −0.0006.

Figure 4

Three-dimensional structures of the heptameric complex (a) 9,028 untilted particle images were grouped by hierarchical ascendant classification and the two depicted classes, comprising 497 and 608 particles, respectively, were chosen for random-conical tilt reconstruction (RCT). (b) Initial maps obtained by RCT from the classes shown in (a) are depicted as isodensity contour surfaces that were low-pass filtered beyond the reproducible resolution (Fourier shell correlation (FSC) = 0.5 at 1/58 Å⁻¹, see Supplementary Fig. 3). (c) The initial maps were used as references for projection matching of all 9,028 tilted particle images (see text for details). 4,430 particles aligned to initial map 1, and 4,598 particles aligned to initial map 2. Final maps 1 and 2 were obtained by the simultaneous iterative reconstruction technique, and are shown in two views as isodensity contour surfaces low-pass filtered beyond the reproducible resolution (FSC = 0.5 at 1/35 Å⁻¹, see Supplementary Fig. 3). All structures depicted to scale; scale bar, 100 Å.

Figure 5

Mapping of nup localization. Heptameric complexes were purified from yeast strains in which one protein of the subcomplex was genomically tagged with green fluorescent protein (GFP): the C terminus of Seh1 (first row), or the C terminus of Nup133 (second row). Aligned class averages of untagged and GFP-tagged particles are shown in columns (1) and (2). The significance map column (3) shows extra density for the GFP-tagged particles above the five-fold pixel-based standard deviation of the class averages. Column (4) shows an overlay of columns (1) and (3).

Figure 6

Protein arrangement within the heptameric complex (a) Segmentation of the particle (map 2) based on mapped nup localizations and previously established biochemical interactions. The particle surface is color-coded to represent the regions of the particle corresponding to different modules. Boundaries between regions are approximate. Scale bar, 100 Å. (b) Docking of available crystal structures (ribbon representation) into map 2 (isodensity contour mesh representation). Two views related by a 90° rotation around a vertical axis are shown. The crystal structures are of: yeast Nup85 (amino acids 1-570 of 744, dark blue) in complex with yeast Seh1 (full length, light blue), yeast Nup145C (amino acids 125-555 of 711, dark green) in complex with human Sec13 (amino acids 1-316 of 322, light green), human Nup107 (the homologue of yeast Nup84, amino acids 658-925 of 925, orange) in complex with human Nup133 (amino acids 934-1156 of 1156, red), and Nup133 (amino acids 76-478 of 1156, red). The conformation of the Nup107·Nup133 fragment is likely to differ from the actual Nup84·Nup133 conformation in map 2, as evidenced by the poor fit, and the structure is included for illustrative purposes only. Empty regions in the particle map correspond to proteins and protein domains for which no crystal structure is available yet. Grey boxes indicate the regions of the map shown in the subsequent panels. (c-e) Detailed views of crystal structures docked into map 2; N and C termini of the crystallized nup domains are indicated.