Structure, Dynamics, Evolution, and Function of a Major Scaffold Component in the Nuclear Pore Complex

Abstract

Summary The nuclear pore complex, composed of proteins termed nucleoporins (Nups), is responsible for nucleocytoplasmic transport in eukaryotes. Nuclear pore complexes (NPCs) form an annular structure composed of the nuclear ring, cytoplasmic ring, a membrane ring, and two inner rings. Nup192 is a major component of the NPC’s inner ring. We report the crystal structure of Saccharomyces cerevisiae Nup192 residues 2–960 [ScNup192(2–960)], which adopts an α-helical fold with three domains (i.e., D1, D2, and D3). Small angle X-ray scattering and electron microscopy (EM) studies reveal that ScNup192(2–960) could undergo long-range transition between “open” and “closed” conformations. We obtained a structural model of full-length ScNup192 based on EM, the structure of ScNup192(2–960), and homology modeling. Evolutionary analyses using the ScNup192(2–960) structure suggest that NPCs and vesicle-coating complexes are descended from a common membrane-coating ancestral complex. We show that suppression of Nup192 expression leads to compromised nuclear transport and hypothesize a role for Nup192 in modulating the permeability of the NPC central channel.

Integrative structure and functional anatomy of a nuclear pore complex

“…SAXS measurements for 147 constructs of 18 Nups 43,72,88–92 (Supplementary Table 6; manuscript in preparation; source data are provided with this article) were carried out both at the Stanford Synchrotron Radiation Lightsource Beamline 4-2 in the SLAC National Accelerator Laboratory (Menlo Park, CA) and at the SIBYLS Beamline 12.3.1 of the Advanced Light Source in the Lawrence Berkeley National Laboratory (Berkeley, CA). SAXS data were collected at concentrations ranging from 0.5 to 5.0 (or higher, depending on the sample) mg/mL, using the previously defined standard protocol 43,88,90 ; ~20 one-second exposures were used for each sample and buffers maintained at 15°C.…”

“…Representations of individual Nups and some of their subcomplexes (Supplementary Table 2 and references therein) relied on (1) atomic structures of 21 yeast Nup domains and 3 sub-complexes determined by X-ray crystallography or NMR spectroscopy; (2) our structures of Nup116, Nup133, Nup145N, Nup192, and Pom152, as well as the Nup82 and Nup84 sub-complexes solved by integrative structure determination 43,66,72,88–92 ; (3) 29 comparative models built with MODELLER 9.13 108 based on the known structure(s) detected by HHPred 109,110 and the literature; (4) SAXS profiles for 147 constructs of 18 Nups 43,72,88–92 (Supplementary Table 6; manuscript in preparation); (5) secondary structure, disordered regions, and domain boundaries predicted by PSIPRED 111,112 , DISOPRED 113 , and DomPred 114 , respectively; (6) coiled-coil regions of Nup82, Nup159, Nsp1, Nup49, Nup57, Mlp1, and Mlp2 predicted by COILS/PCOILS 115 and Multicoil2 116 ; (7) an atomic structure of the Nup53 229–365 RRM domain from S. cerevisiae determined by X-ray crystallography (manuscript in preparation); and (8) the negative-stain EM density maps of full-length Nup192 (EMD-5556 92 ) and Pom152 (EMD-8543 43 ). See Supplementary Table 2 and references therein.…”

“…We used the SAXS data to confirm the rigid-body representations of 8 Nups with X-ray structures, comparative models, and previously published atomic integrative structures 43,72,88–92 (Supplementary Tables 2 and 6; Extended Data Fig. 7F).…”

“…7F). The rigid-body representation of a Nup construct was validated by a chi-value that quantifies the difference between the computed (from an atomic rigid-body representation using FoXS 119 ) and experimental SAXS profiles, except for several constructs of Nup133 and Nup192 that were flexible during integrative modeling and were thus evaluated as described in detail in our original publications 90,92,120 . The chi-value validation assumes that each Nup construct, corresponding in most cases to a single domain (not the whole protein), has the same conformation in solution and in complex; this assumption is consistent with other data ( e.g.…”

Comparative interactomics provides evidence for functional specialization of the nuclear pore complex

“…18 Furthermore, the 2 large yeast inner ring a-solenoid ScNups188/192 (Table 1) share architectural features with karyopherins (also a-solenoid proteins), suggestive of a co-evolution of transport factors, structural components of the NPC, and the endomembrane trafficking system. 72,73 Indeed, it is noteworthy that the assembly of vesicle coats and the translocation of proteins through membranes are controlled by Ras-like GTPases termed Rabs, 74 just as the translocation of cargo in the NPC is controlled by the Ras-like GTPase, Ran.…”