Architecture of the cytoplasmic face of the nuclear pore

Abstract

INTRODUCTION The subcellular compartmentalization of eukaryotic cells requires selective transport of folded proteins and protein-nucleic acid complexes. Embedded in nuclear envelope pores, which are generated by the circumscribed fusion of the inner and outer nuclear membranes, nuclear pore complexes (NPCs) are the sole bidirectional gateways for nucleocytoplasmic transport. The ~110-MDa human NPC is an ~1000-protein assembly that comprises multiple copies of ~34 different proteins, collectively termed nucleoporins. The symmetric core of the NPC is composed of an inner ring encircling the central transport channel and outer rings formed by Y‑shaped coat nucleoporin complexes (CNCs) anchored atop both sides of the nuclear envelope. The outer rings are decorated with compartment‑specific asymmetric nuclear basket and cytoplasmic filament nucleoporins, which establish transport directionality and provide docking sites for transport factors and the small guanosine triphosphatase Ran. The cytoplasmic filament nucleoporins also play an essential role in the irreversible remodeling of messenger ribonucleoprotein particles (mRNPs) as they exit the central transport channel. Unsurprisingly, the NPC's cytoplasmic face represents a hotspot for disease‑associated mutations and is commonly targeted by viral virulence factors. RATIONALE Previous studies established a near-atomic composite structure of the human NPC's symmetric core by combining (i) biochemical reconstitution to elucidate the interaction network between symmetric nucleoporins, (ii) crystal and single-particle cryo-electron microscopy structure determination of nucleoporins and nucleoporin complexes to reveal their three-dimensional shape and the molecular details of their interactions, (iii) quantitative docking in cryo-electron tomography (cryo-ET) maps of the intact human NPC to uncover nucleoporin stoichiometry and positioning, and (iv) cell‑based assays to validate the physiological relevance of the biochemical and structural findings. In this work, we extended our approach to the cytoplasmic filament nucleoporins to reveal the near-atomic architecture of the cytoplasmic face of the human NPC. RESULTS Using biochemical reconstitution, we elucidated the protein-protein and protein-RNA interaction networks of the human and Chaetomium thermophilum cytoplasmic filament nucleoporins, establishing an evolutionarily conserved heterohexameric cytoplasmic filament nucleoporin complex (CFNC) held together by a central heterotrimeric coiled‑coil hub that tethers two separate mRNP‑remodeling complexes. Further biochemical analysis and determination of a series of crystal structures revealed that the metazoan‑specific cytoplasmic filament nucleoporin NUP358 is composed of 16 distinct domains, including an N‑terminal S‑shaped α‑helical solenoid followed by a coiled‑coil oligomerization element, numerous Ran‑interacting domains, an E3 ligase domain, and a C‑terminal prolyl‑isomerase domain. Physiologically validated quantitative docking into cryo-ET maps of the intact human NPC revealed that pentameric NUP358 bundles, conjoined by the oligomerization element, are anchored through their N‑terminal domains to the central stalk regions of the CNC, projecting flexibly attached domains as far as ~600 Å into the cytoplasm. Using cell‑based assays, we demonstrated that NUP358 is dispensable for the architectural integrity of the assembled interphase NPC and RNA export but is required for efficient translation. After NUP358 assignment, the remaining 4-shaped cryo‑ET density matched the dimensions of the CFNC coiled‑coil hub, in close proximity to an outer-ring NUP93. Whereas the N-terminal NUP93 assembly sensor motif anchors the properly assembled related coiled‑coil channel nucleoporin heterotrimer to the inner ring, biochemical reconstitution confirmed that the NUP93 assembly sensor is reused in anchoring the CFNC to the cytoplasmic face of the human NPC. By contrast, two C. thermophilum CFNCs are anchored by a divergent mechanism that involves assembly sensors located in unstructured portions of two CNC nucleoporins. Whereas unassigned cryo‑ET density occupies the NUP358 and CFNC binding sites on the nuclear face, docking of the nuclear basket component ELYS established that the equivalent position on the cytoplasmic face is unoccupied, suggesting that mechanisms other than steric competition promote asymmetric distribution of nucleoporins. CONCLUSION We have substantially advanced the biochemical and structural characterization of the asymmetric nucleoporins' architecture and attachment at the cytoplasmic and nuclear faces of the NPC. Our near‑atomic composite structure of the human NPC's cytoplasmic face provides a biochemical and structural framework for elucidating the molecular basis of mRNP remodeling, viral virulence factor interference with NPC function, and the underlying mechanisms of nucleoporin diseases at the cytoplasmic face of the NPC. [Figure: see text].

Fig. 0.. Cytoplasmic face of the human nuclear pore complex (NPC).

Near-atomic composite structure of the NPC generated by docking high-resolution crystal structures into a cryo-ET reconstruction of an intact human NPC. The symmetric core, embedded in the nuclear envelope, is decorated with NUP358 (red) domains bound to Ran (gray) flexibly projected into the cytoplasm and cytoplasmic filament nucleoporin complexes (pink) overlooking the central transport channel.

Fig. 1.. Reconstitution of a 16-protein C.thermophilum coat-cytoplasmic filament nup complex.

(A) Cross sectional schematic of the fungal NPC architecture. (B) Domain structures of the coat and cytoplasmic filament nups. (C) Schematic representation summarizing our biochemical reconstitution and dissection experiments with purified recombinant C.thermophilum nups, illustrating the cytoplasmic filament nup complex (CFNC) architecture and its attachment to the coat nup complex (CNC). The CNC harbors two assembly sensors, Nup37^CTE and Nup145C^NTE, each anchoring a CFNC via its central hub, with Nup37^CTE exhibiting tighter binding than Nup145C^NTE. (D-F) SEC-MALS interaction analyses, showing the stepwise biochemical reconstitution starting with (D) the CFNC (green) from Nup82•Nup159•Nsp1 (blue), Gle2•Nup145N (cyan), and Dbp5 (red), (E) CFNC•Gle1•Nup42^GBM (green) from CFNC (blue) and Gle1•Nup42^GBM (red), and culminating with (F) the 16-protein CNC•CFNC•Gle1•Nup42^GBM complex (green) from CNC (red), CFNC (blue), and Gle1•Nup42^GBM (cyan). SDS-PAGE gel strips of peak fractions are shown. Measured molecular masses are indicated, with respective theoretical masses in parentheses. (G, H) Liquid-liquid phase separation (LLPS) interaction assays, assessing (G) CFNC (red) and Gle1•Nup42^GBM (cyan) incorporation into CNC-LLPS (green), and (H) CFNC incorporation into CNC-LLPS, lacking either one or both Nup37^CTE and Nup145C^NTE assembly sensors. N-terminally fluorescently labeled CNC (Bodipy), CFNC (Alexa Fluor 647), and Gle1•Nup42^GBM (Coumarin) were visualized by fluorescence microscopy. Pelleted CNC condensate phase (P) and soluble (S) fractions were analyzed by SDS-PAGE and visualized by Coomassie brilliant blue staining. Scale bars are 10μm.

Fig. 2.. Conserved modular architecture and RNA binding properties of the human CF nups

(A) Cross sectional schematic of the human NPC architecture. (B) Domain structures of human cytoplasmic filament nups. Nomenclature of H.sapiens and C.thermophilum nup orthologs is indicated. (C) Biochemical reconstitution of the ~310kDa hetero-hexameric human CFNC. SEC-MALS interaction analysis of NUP88•NUP214•NUP62 (blue), DDX19 (ADP) (red), RAE1•NUP98 (cyan), and their preincubation (green). Measured molecular masses are indicated, with theoretical masses in parentheses. SDS-PAGE gel strips of peak fractions visualized by Coomassie brilliant blue staining are shown. (D) Summary of pairwise SEC-MALS interaction analyses between human cytoplasmic filament nups. (E) Schematic summary of the human CFNC architecture and the cytoplasmic filament nup interaction network. (F) Human cytoplasmic filament nup domains and complexes were assayed for binding to single-stranded (ss) and double-stranded (ds) RNA and DNA probes by electrophoretic mobility shift assay (EMSA). Input proteins resolved by SDS-PAGE were visualized by Coomassie brilliant blue staining. Qualitative assessment of nucleic acid binding is denoted by color-coded boxes. (G, H) EMSAs with ssRNA titrated against (G) metazoan-specific NUP358^NTD and NUP358^RanBD-IV•Ran(GMPPNP), and (H) the indicated H.sapiens CFNC subcomplexes and their C.thermophilum orthologs.

Fig. 3.. Structural analysis and biochemical characterization of NUP358.

(A) Domain structure of NUP358. Black lines indicate the boundaries of the crystallized fragments. (B, C) SEC-MALS analysis of the oligomeric behavior of (B) NUP358^NTD and NUP358^NTDΔTPR, and (C) NUP358^NTD-OE, performed at the indicated protein concentrations. Measured molecular masses are indicated, with theoretical masses in parentheses. SDS-PAGE gel strips of peak fractions are shown and visualized by Coomassie brilliant blue staining. (D) Cartoon representation of the NUP358^NTD•sAB-14 co-crystal structure dyad of the P6₅22 lattice, illustrating the NUP358^NTD dimer between symmetry-related molecules. (E) Schematic of the NUP358^NTD structure and structural motifs. (F) TPR of molecule 1 complements α-helical stacking of the N-terminal solenoid of the symmetry-related molecule 2, generating the open conformation of NUP358^NTD. (G) SEC-MALS analysis of the oligomeric behavior of NUP358^OE and the NUP358^OE LIQIML mutant performed at the indicated protein concentrations. (H) Cartoon representation of the homo-tetrameric NUP358^OE crystal structure with hydrophobic core residues shown in ball-and-stick representation. (I) SEC interaction analysis of NUP358^ZnF7 and NUP358^ZnF7ΔNTE binding to Ran(GTP). (J) SEC-MALS interaction analysis of NUP358^RanBD-IV binding to Ran(GMPPNP). (K) Co-crystal structure of NUP358^ZnF7•Ran(GDP), shown in cartoon and surface representation (left). The inset indicates the location of the magnified and 90° rotated view of the Ran hydrophobic pocket (middle). Superposition of the six NUP358^ZnF•Ran(GDP) and four NUP153^ZnF•Ran(GDP) co-crystal structures with the Zn²⁺-coordinating cysteines and Ran-burying NTE hydrophobic residues shown as sticks (right). (L) Co-crystal structure of NUP358^RanBD-IV•Ran(GMPPNP) with NUP358^RanBD-IV shown in cartoon and Ran(GMPPNP) shown in cartoon (left) or surface (middle) representation. Superposition of Ran(GMPPNP) bound to NUP358^RanBD-I, NUP358^RanBD-II, NUP358^RanBD-III, NUP358^RanBD-IV, and NUP50^RanBD (right).

Fig. 4.. Docking of NUP358^NTD on the cytoplasmic face of the NPC.

(A) Overview of the NPC cytoplasmic face with isosurface rendering of unexplained density clusters I (red) and II (cyan) of the ~12Å cryo-ET map of the intact human NPC. The inset indicates the location of the magnified view showing cartoon representations of five copies of NUP358^NTD docked in unassigned density cluster I. (B) Comparison of the binding of two NUP358^NTD copies (cartoon) to distal and proximal CNCs (surface). (C-E) Architecture of the pentameric NUP358^NTD bundle attachment site on a cytoplasmic outer ring spoke with cartoon- and surface-represented structures (left) and schematics (right), sequentially illustrating the placement of (C) four copies of NUP358^NTD around NUP96 and NUP107 interfaces on the stalks of tandem-arranged Y-shaped CNCs, (D) a distal copy of NUP93^SOL collocated at the center of the NUP358^NTD bundle, interfacing with both proximal and distal CNC stalks, and (E) the NUP358^NTD dome copy interfacing with the stalk-attached NUP358^NTD quartet beneath. (F) Overview of the entire cytoplasmic face of the NPC in cartoon representation and as schematic, illustrating the distribution of 40 NUP358^NTD copies anchored as pentameric bundles across the eight NPC spokes. (G) Schematic of NUP358 attachment to the cytoplasmic outer ring spoke. The NUP358 pentameric bundle is linked together by interactions between oligomerization elements (OEs). Anchored by NUP358^NTD, the rest of NUP358 domains are linked by unstructured linker sequences and are expected to freely project from the cytoplasmic face of the NPC. Distal and proximal positions are labeled according to the legend in (A).

Fig. 5.. NUP358 plays a general role in translation of exported mRNA.

(A) Subcellular localization by immunofluorescent staining of endogenous nups in synchronized AID::NUP358 HCT116 cells at the indicated time points following auxin-induced NUP358 depletion. Cytoplasmic puncta arise from NUP88 overexpression inherent to HCT116 cells. (B) Subcellular localization of N-terminally 3×HA-tagged NUP358 variants in AID::NUP358 HCT116 cells by immunofluorescence microscopy. mAb414 staining marks nuclear envelope rim location. Domain structure of the transfected NUP358 variants is shown (above). (C) Schematic illustrating the life cycle of messenger ribonucleoprotein particles (mRNPs) from transcription, maturation, export, remodeling at the cytoplasmic face of the NPC, to translation. Steps associated with the NPC are highlighted by a red box. (D) Time-resolved analysis of RNA nuclear retention in synchronized AID::NUP358 HCT116 cells upon auxin-induced NUP358 depletion visualizing 5-EU metabolically pulse-labeled RNA. Representative images (left) and quantitation (right) of the proportion of cells (n>200/timepoint) with nuclear RNA retention are shown with mean values (squares) and the respective standard error (shaded area) of triplicate experiments. Quantitation from unsynchronized AID::NUP358 DLD1 and NUP160::NG-AID DLD1 cells are also shown. (E) Time-resolved western blot analysis of the expression of C-terminally 3×FLAG-tagged reporter proteins in synchronized AID::NUP358 HCT116 cells upon auxin-induced NUP358 depletion. Quantitated reporter expression in auxin-treated cells was normalized to expression in control cells, at the 10-hour timepoint. Experiments performed in triplicate, with mean and associated standard error shown. Scale bars are 10μm. Experimental timelines are shown above each experiment.

Fig. 6.. Docking analysis reveals that NUP93 anchors the CFNC to the cytoplasmic outer ring.

(A) Overview of the NPC cytoplasmic face with isosurface rendering of unexplained density cluster II (cyan) of the ~12Å cryo-ET map of the intact human NPC. The inset indicates the location of the magnified view in (B). (B, C) Two views of manually placed poly-alanine models of CFNC-hub segments CCS1 and CCS2, as well as of the tentatively placed NUP88^NTD•NUP98^APD and NUP214^NTD•DDX19 co-crystal structures shown in cartoon representation. (D) Cartoon representation of a cytoplasmic face spoke. The root-mean square (r.m.s.) end-to-end length estimate for the NUP93^R1-R2 linker defines a ~40Å radius for the expected location of the distal NUP93^R1 region. (E) Schematic of a cytoplasmic face spoke illustrating CFNC-hub anchoring by the distal NUP93^R1 positioned by the distal NUP205-bound NUP93^R2. (F) SEC-MALS interaction analysis showing the binding of SUMO-NUP93^R1 to the hetero-hexameric CFNC or CFNC-hub and illustrating the abolished binding of the SUMO-NUP93^R1 LIL mutant to the CFNC-hub. Proteins were visualized by Coomassie brilliant blue staining. (G) An alignment of C.thermophilum Nic96 and H.sapiens NUP93 sequences shows the conservation of residues targeted by the LLLL and LIL mutations that abolish binding to the CNT. Residues are colored according to a multispecies sequence alignment from white (less than 55% similarity), to yellow (55% similarity), to red (100% identity), using the BLOSUM62 weighting algorithm. Domain architectures of NUP214, NUP88, NUP62, and NUP93. The location of the NUP93 LIL mutation is indicated (red dots). (H) Two views of the NUP88^NTD•NUP98^APD co-crystal structure in cartoon representation. Inset boxes indicate regions of magnified views (right) of NUP88^NTD FGL-loop interaction with NUP98^APD, and of NUP98^APD K/R-loop interaction with NUP88^NTD. Black triangles indicate alanine substitutions in the NUP88^NTD EMNY mutant.

Fig. 7.. Comparison of cytoplasmic and nuclear faces of the human NPC.

Overall top view (left), single spoke protomer with symmetric core nups in surface, docked asymmetric nups in cartoon, and unexplained density of the ~12Å cryo-ET map in isosurface representation (middle), and schematic (right) of the (A) cytoplasmic and (B) nuclear face of the intact human NPC. (C) Superpositions of the overall view (left) and two orthogonal views of single spoke protomers (middle and right) of the nuclear and cytoplasmic faces, with hypothetical steric clashes between the cytoplasmic filaments in cartoon representation, and the unassigned asymmetric nuclear density (cyan), indicated. Distal and proximal positions are labeled according to the legend. Inset boxes indicate regions of magnified protomer views (right).

Fig. 8.. Architecture of the human NPC cytoplasmic face.

Near-atomic composite structure of the human NPC generated by docking individual nup and nup complex crystal and cryo-EM structures into a ~12Å cryo-ET map of the intact human NPC, viewed from (A) the cytoplasmic face, and (B) the central transport channel, as a cross-section. Newly placed structures include the quantitatively docked NUP358^NTD and the manually docked NUP88^NTD•NUP98^APD, NUP214^NTD•DDX19, GLE1^CTD•NUP42^GBM, ELYS^NTD, and a CFNC-hub model. The nuclear envelope is rendered as a grey isosurface. Nups are shown in cartoon representation and colored according to the legend.