- アルゴンヌの先端光源での20年近い研究の結果、研究者は原子核の「門番」の姿を明らかにし、その仕組みについてより良い考えを持つに至った。 After nearly 20 years of work, much of it at Argonne’s Advanced Photon Source, researchers have a clearer picture of the “gatekeeper” of the nucleus and a better idea of how it works.
アルゴンヌの先端光源での20年近い研究の結果、研究者は原子核の「門番」の姿を明らかにし、その仕組みについてより良い考えを持つに至った。 After nearly 20 years of work, much of it at Argonne’s Advanced Photon Source, researchers have a clearer picture of the “gatekeeper” of the nucleus and a better idea of how it works.
Architecture of the cytoplasmic face of the nuclear pore
Christopher J. Bley,Si Nie,George W. Mobbs,Stefan Petrovic,Anna T. Gres,Xiaoyu Liu,Somnath Mukherjee,Sho Harvey,Ferdinand M. Huber,Daniel H. Lin ,Bonnie Brown,Aaron W. Tang,Emily J. Rundlet,Ana R. Correia,Shane Chen ,Saroj G. Regmi,Taylor A. Stevens ,Claudia A. Jette ,Mary Dasso ,Alina Patke ,Alexander F. Palazzo ,Anthony A. Kossiakoff,André Hoelz
Science Published:10 Jun 2022
Cytoplasmic face of the human 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 CFNCs (pink) overlooking the central transport channel.
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.
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.
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.
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.
The nuclear pore complex (NPC) is the sole bidirectional gateway for nucleocytoplasmic transport. Despite recent progress in elucidating the NPC symmetric core architecture, the asymmetrically decorated cytoplasmic face, essential for messenger RNA (mRNA) export and a hotspot for nucleoporin-associated diseases, has remained elusive. Here we report a composite structure of the human cytoplasmic face obtained by combining biochemical reconstitution, crystal structure determination, docking into cryo–electron tomographic reconstructions, and physiological validation. Whereas species-specific motifs anchor an evolutionarily conserved ~540-kilodalton heterohexameric cytoplasmic filament nucleoporin complex above the central transport channel, attachment of the NUP358 pentameric bundles depends on the double-ring arrangement of the coat nucleoporin complex. Our composite structure and its predictive power provide a rich foundation for elucidating the molecular basis of mRNA export and nucleoporin diseases.
核膜孔のリンカー・スキャフォールドの構造 Architecture of the linker-scaffold in the nuclear pore
Stefan Petrovic ,Dipanjan Samanta,Thibaud Perriches,Christopher J. Bley ,Karsten Thierbach,Bonnie Brown,Si Nie ,George W. Mobbs ,Taylor A. Stevens,Xiaoyu Liu,Giovani Pinton Tomaleri,Lucas Schaus ,André Hoelz
Science Published:10 Jun 2022
Linker-scaffold architecture in the human NPC’s symmetric core.
Near‑atomic composite structure of the NPC’s symmetric core obtained by quantitative docking of high-resolution crystal and single-particle cryo-EM structures into a cryo-ET reconstruction of the intact human NPC. Schematic representations of the intricate linker-scaffold topology of the cytoplasmic outer ring, inner ring, and nuclear outer ring (clockwise from top) are depicted for the boxed regions. C, C terminus; N, N terminus.
In eukaryotic cells, the selective bidirectional transport of macromolecules between the nucleus and cytoplasm occurs through the nuclear pore complex (NPC). Embedded in nuclear envelope pores, the ~110-MDa human NPC is an ~1200-Å-wide and ~750-Å-tall assembly of ~1000 proteins, collectively termed nucleoporins. Because of the NPC’s eightfold rotational symmetry along the nucleocytoplasmic axis, each of the ~34 different nucleoporins occurs in multiples of eight. Architecturally, the NPC’s symmetric core is composed of an inner ring encircling the central transport channel and two outer rings anchored on both sides of the nuclear envelope. Because of its central role in the flow of genetic information from DNA to RNA to protein, the NPC is commonly targeted in viral infections and its nucleoporin constituents are associated with a plethora of diseases.
Although the arrangement of most scaffold nucleoporins in the NPC’s symmetric core was determined by quantitative docking of crystal structures into cryo–electron tomographic (cryo-ET) maps of intact NPCs, the topology and molecular details of their cohesion by multivalent linker nucleoporins have remained elusive. Recently, in situ cryo-ET reconstructions of NPCs from various species have indicated that the NPC’s inner ring is capable of reversible constriction and dilation in response to variations in nuclear envelope membrane tension, thereby modulating the diameter of the central transport channel by ~200 Å. We combined biochemical reconstitution, high-resolution crystal and single-particle cryo–electron microscopy (cryo-EM) structure determination, docking into cryo-ET maps, and physiological validation to elucidate the molecular architecture of the linker-scaffold interaction network that not only is essential for the NPC’s integrity but also confers the plasticity and robustness necessary to allow and withstand such large-scale conformational changes.
By biochemically mapping scaffold-binding regions of all fungal and human linker nucleoporins and determining crystal and single-particle cryo-EM structures of linker-scaffold complexes, we completed the characterization of the biochemically tractable linker-scaffold network and established its evolutionary conservation, despite considerable sequence divergence. We determined a series of crystal and single-particle cryo-EM structures of the intact Nup188 and Nup192 scaffold hubs bound to their Nic96, Nup145N, and Nup53 linker nucleoporin binding regions, revealing that both proteins form distinct question mark–shaped keystones of two evolutionarily conserved hetero‑octameric inner ring complexes. Linkers bind to scaffold surface pockets through short defined motifs, with flanking regions commonly forming additional disperse interactions that reinforce the binding. Using a structure‑guided functional analysis in Saccharomyces cerevisiae, we confirmed the robustness of linker‑scaffold interactions and established the physiological relevance of our biochemical and structural findings. The near-atomic composite structures resulting from quantitative docking of experimental structures into human and S. cerevisiae cryo-ET maps of constricted and dilated NPCs structurally disambiguated the positioning of the Nup188 and Nup192 hubs in the intact fungal and human NPC and revealed the topology of the linker-scaffold network. The linker-scaffold gives rise to eight relatively rigid inner ring spokes that are flexibly interconnected to allow for the formation of lateral channels. Unexpectedly, we uncovered that linker‑scaffold interactions play an opposing role in the outer rings by forming tight cross-link staples between the eight nuclear and cytoplasmic outer ring spokes, thereby limiting the dilatory movements to the inner ring.
We have substantially advanced the structural and biochemical characterization of the symmetric core of the S. cerevisiae and human NPCs and determined near-atomic composite structures. The composite structures uncover the molecular mechanism by which the evolutionarily conserved linker‑scaffold establishes the NPC’s integrity while simultaneously allowing for the observed plasticity of the central transport channel. The composite structures are roadmaps for the mechanistic dissection of NPC assembly and disassembly, the etiology of NPC‑associated diseases, the role of NPC dilation in nucleocytoplasmic transport of soluble and integral membrane protein cargos, and the anchoring of asymmetric nucleoporins.
Nuclear pore complexes (NPCs) mediate the nucleocytoplasmic transport of macromolecules. Although the arrangement of the structured scaffold nucleoporins in the NPC’s symmetric core has been determined, their cohesion by multivalent unstructured linker nucleoporins has remained elusive. Combining biochemical reconstitution, high-resolution structure determination, docking into cryo–electron tomographic reconstructions, and physiological validation, we elucidated the architecture of the evolutionarily conserved linker-scaffold, yielding a near-atomic composite structure of the human NPC’s ~64-megadalton symmetric core. Whereas linkers generally play a rigidifying role, the linker-scaffold of the NPC provides the plasticity and robustness necessary for the reversible constriction and dilation of its central transport channel and the emergence of lateral channels. Our results substantially advance the structural characterization of the NPC symmetric core, providing a basis for future functional studies.