The nuclear pore complex encloses a central channel for nucleocytoplasmic transport, which is thought to consist of three nucleoporins, Nup54, Nup58, and Nup62. However, the structure and composition of the channel are elusive. We determined the crystal structures of the interacting domains between these nucleoporins and pieced together the molecular architecture of the mammalian transport channel. Located in the channel midplane is a flexible Nup54⋅Nup58 ring that can undergo large rearrangements yielding diameter changes from ∼20 to ∼40 nm. Nup62⋅Nup54 triple helices project alternately up and down from either side of the midplane ring and form nucleoplasmic and cytoplasmic entries. The channel consists of as many as 224 copies of the three nucleoporins, amounting to a molar mass of 12.3 MDa and contributing 256 phenylalanine-glycine repeat regions. We propose that the occupancy of these repeat regions with transport receptors modulates ring diameter and transport activity.
The nucleoporins Nup58 and Nup45 are part of the central transport channel of the nuclear pore complex, which is thought to have a flexible diameter. In the crystal structure of an alpha-helical region of mammalian Nup58/45, we identified distinct tetramers, each consisting of two antiparallel hairpin dimers. The intradimeric interface is hydrophobic, whereas dimer-dimer association occurs through large hydrophilic residues. These residues are laterally displaced in various tetramer conformations, which suggests an intermolecular sliding by 11 angstroms. We propose that circumferential sliding plays a role in adjusting the diameter of the central transport channel.
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 phenylalanineglycine regions ("FG repeats") of channel nups. Akin to ligandgated 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.ligand gating | X-ray crystallography | nucleo-cytoplasmic transport I n evolution from prokaryotes to eukaryotes, the myriad of reactions yielding cotranscriptional and posttranscriptional assembly of ribonucleoproteins was largely confined to the nuclear compartment. This required the concomitant evolution of transport conduits of a sufficiently large diameter to allow passage of assembled ribonucleoproteins to the cytoplasm. Among these, newly assembled ribosomal subunits presented a special challenge, as they are relatively rigid bodies; the eukaryotic large ribosomal subunit, for example, has a diameter of about 30 nm. Hence, a correspondingly large nuclear pore had to evolve to accommodate the passage of ribosomal subunits, but also to protect against concomitant bidirectional leakage of the myriad of proteins, which are generally much smaller than ribonucleoproteins, to help maintain distinct nucleoplasmic and cytoplasmic proteomes. One way for meeting the challenge to transport diverse substrates of a large size range would be to endow the nuclear pore with the ability to have its central transport channel undergo dilation and constriction. However, as has been argued elsewhere (1), diameter changes in the 30-nm range might buckle the membrane, if channel proteins were directly embedded into the membrane's lipid bilayer. It could be conjectured that evolution of the nuclear pore into the massive protein...
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