Nuclear pores. Architecture of the nuclear pore complex coat

Abstract

The nuclear pore complex (NPC) constitutes the sole gateway for bidirectional nucleocytoplasmic transport. Despite half a century of structural characterization, the architecture of the NPC remains unknown. Here we present the crystal structure of a reconstituted ~400-kilodalton coat nucleoporin complex (CNC) from Saccharomyces cerevisiae at a 7.4 angstrom resolution. The crystal structure revealed a curved Y-shaped architecture and the molecular details of the coat nucleoporin interactions forming the central "triskelion" of the Y. A structural comparison of the yeast CNC with an electron microscopy reconstruction of its human counterpart suggested the evolutionary conservation of the elucidated architecture. Moreover, 32 copies of the CNC crystal structure docked readily into a cryoelectron tomographic reconstruction of the fully assembled human NPC, thereby accounting for ~16 megadalton of its mass.

Fig. 1. Overall architecture of the CNC

(A) Domain structures of the yeast coat nups and sAB-57. Black lines indicate the crystallized fragments. U: unstructured, D: domain invasion motif, VH: heavy chain variable region, CH: heavy chain constant region, VL: light chain variable region, CL: light chain constant region. (B) Reconstitution of the yeast CNC•sAB-57, lacking Nup133. Elution profiles from a Superdex 200 10/300 column are shown for Nup120•Seh1•Nup85 (Trimer 1), Sec13•Nup145C•Nup84^NTD (Trimer 2), CNC, and CNC•sAB-57 (left). SDS-PAGE gel of the reconstituted CNC•sAB-57 used for crystallization (right). (C) Cartoon and schematic representations of the yeast CNC•sAB-57 crystal structure viewed from two sides.

Fig. 2. Architecture of the CNC triskelion

Cartoon representation of the triskelion formed by Nup120, Nup85 and Nup145C. Insets (A–C) depict magnified views for the interactions between (A) Nup120^CTD, Nup85^CTD, and Nup145C^CTD (B) Nup120^CTD, Nup85^CTD, and N-terminal Nup145C helix; and (C) Nup145C, Nup85^CTD, and sAB-57. The density modified electron density map is contoured at 1.0 σ.

Fig. 3. Comparison of yeast and human CNCs

(A) Fit of the yeast CNC crystal structure into the human CNC negative-stain EM reconstruction (gray) (3). Arrows indicate density accounted for by the additional human coat nups Nup37 or Nup43. (B) Comparison of the quality of fit for the yeast CNC crystal structure and human CNC EM reconstruction (cyan) into the intact human NPC cryoelectron tomographic reconstruction (gray) (3). Arrows indicate regions where the human CNC EM reconstruction protrudes from the cryoelectron tomographic reconstruction.

Fig. 4. Architecture of the NPC coat

(A) 32 copies of the yeast CNC, shown in cartoon representation with a representative subunit colored as in Fig. 1, docked into the cryoelectron tomographic reconstruction of the intact human NPC (3), shown as a gray surface. The outer and inner cytoplasmic and nuclear CNC rings are highlighted in orange, cyan, pink, and blue, respectively. (B) Cartoon representations of 16 yeast CNC copies from the cytoplasmic side of the NPC coat. Schematics indicating the positions assigned to Nup84^CTD and Nup133, which were not crystallized, are shown. (C) Interface between the inner and outer CNC rings. Two views of the yeast CNC and its mate from the inner ring are shown. (D) Orientation of the Nup120 β-propeller relative to neighboring coat nups and the membrane. Portions of two CNCs from the cytoplasmic outer ring are shown in cartoon representation. Green and cyan shading indicates the positioning of Nup84^CTD and Nup133, respectively. The cyan line represents the N-terminal unstructured segment of Nup133 that binds to Nup120 (9). A schematic representation of the ring-forming Nup120-Nup133 interaction is shown below.