Ring cycle for dilating and constricting the nuclear pore

Abstract

We recently showed that the three "channel" nucleoporins, Nup54, Nup58, and Nup62, interact with each other through only four distinct sites and established the crystal structures of the two resulting "interactomes," Nup54•Nup58 and Nup54•Nup62. We also reported instability of the Nup54•Nup58 interactome and previously determined the atomic structure of the relevant Nup58 segment by itself, demonstrating that it forms a twofold symmetric tetramer. Here, we report the crystal structure of the relevant free Nup54 segment and show that it forms a tetrameric, helical bundle that is structurally "conditioned" for instability by a central patch of polar hydrogen-bonded residues. Integrating these data with our previously reported results, we propose a "ring cycle" for dilating and constricting the nuclear pore. In essence, three homooligomeric rings, one consisting of eight modules of Nup58 tetramers, and two, each consisting of eight modules of Nup54 tetramers, are stacked in midplane and characterize a constricted pore of 10- to 20-nm diameter. In going to the dilated state, segments of one Nup58 and two Nup54 tetrameric modules reassort into a dodecameric module, eight of which form a single, heterooligomeric midplane ring, which is flexible in a diameter range of 40-50 nm. The ring cycle would be regulated by phenylalanine-glycine regions ("FG repeats") of channel nups. Akin to ligand-gated channels, the dilated state of the midplane ring may be stabilized by binding of [cargo•transport-factor] complexes to FG repeats, thereby linking the ratio of constricted to dilated nuclear pores to cellular transport need.

Fig. 1.

Interaction domains of channel nups. Domain representation of Nup58 (red), Nup54 (blue), and Nup62 (gray) from Rattus norvegicus indicating α-helical (saturated color, bordering residues are indicated by solid triangles), α/β (labeled), and unstructured regions (light shade of color) with FG repeats (black lines) (11). Mapped interacting domains of each channel nup are marked by a bar in the color of the respective interacting partner and labeled (12). Unsaturated “valences” of Nup58 and Nup62 are marked by a green and yellow bar, respectively. A glycosylation region of Nup62 (see Discussion) is marked by an asterisk (Fig. S1A).

Fig. 2.

Structure of Nup54. (A and B) Cartoon representation of the Nup54 tetramer (blue), related by a 90° rotation. A twofold axis of symmetry is indicated (orange). Helix I and II conformers are located at the top and bottom of the tetramer, respectively. (C) Ribbon representation of Nup54. Residues that mediate hydrophobic (gray) and polar (orange) interactions in the tetramer are shown in van der Waals sphere representation. (D) Cross-section of the tetrameric interface of Nup54 (boxed in C). A small cluster of polar residues, H469 and Q473 (orange; water molecules as pink spheres), forms a dynamic network of polar hydrogen bonds (red or black dashes). Note that H469 can only form one hydrogen bond at a time (black or red dashes). Furthermore, Q473 has two alternative conformations, shown in the upper and lower panels, respectively. (E) Surface representation of the Nup54 tetramer (blue). Residues H469 and Q473 are highlighted in orange and yellow, respectively. See also Fig. S2.

Fig. 3.

CD spectroscopy analysis of Nup54 and its variants. (A) CD wavelength spectra of Nup54 wild-type (wt) (blue) and its variants H469F (green) and H469F/Q473L (red) at 8 °C. The mean residue molar ellipticity [Θ] versus the wavelength is shown. (B) Thermal melting curves of Nup54 wt and its variants H469F and H469F/Q473L, monitored by CD spectroscopy at 222 nm. Unfolding is monitored by the increase of [Θ] as a function of temperature. The resulting melting temperatures T_m are indicated. (C and D) CD wavelength spectra at 8 °C and 100 °C of (C) Nup54 wt (blue, light blue) and (D) Nup54 H469F/Q473L (red, pink).

Fig. 4.

Ring cycle for dilating and constricting the nuclear pore. (A–D) Summary of modules of determined X-ray structures of segments derived from channel nucleoporins Nup54, Nup58, and Nup62 (Fig. 1). (A) Nup54•Nup58 dodecamer consisting of eight Nup54 (blue) and four Nup58 (red) protomers; note the “bent” and “straight” conformers of the Nup54 protomer (12). (B) Nup58 homotetramer; each protomer consists of a helical hairpin (11). (C) Nup54 homotetramer with two distinct conformers of the Nup54 protomer (Fig. S3 A and B). (D) Nup62•Nup54 triple helix (12), with two protomers of Nup62 in gray and one protomer of Nup54 in blue. For visual simplification, the structural elements of the modules are schematically modified in E–J. (E and F) We propose that eight of each of the modules shown in A–C form a ring, representing dilated and constricted states. (E) In the dilated state, eight Nup54•Nup58 dodecamers form a midplane ring, the diameter of which is flexible in the range between 40–50 nm as a result of intramodular sliding (12). (F) For constricting the pore, the 96 constituent protomers of the dilated midplane ring resolve into three homooligomeric rings of 32 protomers each; because of its twofold symmetry, the Nup58 ring is placed in midplane (11), whereas the two Nup54 rings are tentatively placed below and above (F) or tucked within the Nup58 ring (Fig. S5). The three homooligomeric rings, stacked in midplane, display a diameter in the 10- to 20-nm range (Figs. S4 and S5). (G and H) Finger attachments to the dilated midplane ring (G) and to the two Nup54 rings in the constricted state (H): A flexible linker region of Nup54, indicated as blue transparent tube, continues into a Nup62•Nup54 triple helix (D), simplified to a vertically oriented, blue and gray cylinder and termed finger; note the presence of a total of 64 fingers, 32 each on the nucleoplasmic and cytoplasmic sides; cycling from a dilated to a constricted state (G to H) increases crowding of fingers. (I and J) Schematic tracing of full-length channel nups (Fig. 1) in a vertical slice across the dilated (I) and constricted (J) state; distinct nup regions are labeled (see text) and indicated by arrows; color-coded N depicts the N terminus in one each of three channel nups; an “unsaturated valence” of Nup62 (only one of two is indicated) and of Nup58 for a yet-to-be-identified segment of another nup, is indicated by a yellow and a green bar, respectively (Fig. 1); note location of the two FG repeats of Nup58: C-terminal FG repeat projects to the center of the midplane ring, whereas the N-terminal FG repeat projects away from the pore, suggesting that it may bind to a solenoid nucleoporin in the surrounding of the central transport channel. Tracing the path of all three channel nucleoporins, Nup58, Nup54, Nup62, through a single transport channel of the NPC, in copies of 32:64:128 (12), indicates that the three channel nups not only line the channel but also anchor it to the surrounding matrix of nups. Anchorage would be accomplished by 128 binding sites emanating from Nup62, at the base of the fingers, and by 32 binding sites from the Nup58 midplane ring.