Architecture of the linker-scaffold in the nuclear pore

Abstract

INTRODUCTION 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. RATIONALE 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. RESULTS 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. CONCLUSION 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. 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.

A quantitative map of nuclear pore assembly reveals two distinct mechanisms

“…The need for better methods to determine dynamic and variable copy numbers of Nups reliably at the level of single protein complexes under physiological conditions is further highlighted by the fact that our live-cell-based approach resulted in copy number estimates that differ from estimates derived by other approaches for four out of the ten investigated Nups. The main methods used in the field, including quantitative mass spectrometry from cell populations 23 , fitting of single protein structures into highly averaged cryo-EM densities 33 , 34 , as well as calibrated imaging of fluorescently tagged knock-in proteins in single living cells as used in our study, currently all have limitations in this regard. For example, cryo-EM based averaging often selects ‘complete’ NPCs, to obtain a ‘fully occupied’ average NPC model, whereas live-cell imaging data average NPC stoichiometry from all pores in one cell and thus include pore-to-pore variability, which can result in lower estimates.…”

“…8 ). The pseudoatomic model of the native NPC structure 27 , 28 we imposed as the endpoint of the assembly pathway does not include the Y-complex-bound fraction of Nup205 or the Nup214-bound fraction of Nup62, and thus contains only 16 of the 40 copies of Nup205 33 , 34 and 32 of the 48 copies of Nup62 35 in the fully mature NPC. How the remaining 24 copies of Nup205 and 16 copies of Nup62 are assembled remains elusive.…”

Electron microscopy holdings of the Protein Data Bank: the impact of the resolution revolution, new validation tools, and implications for the future

“…The integrative structure of the yeast NPC can be sub-divided into the membrane ring, inner and outer rings, a cytoplasmic export platform, the nuclear basket and the disordered FG repeats that fill the pore. The integrative structure of the yeast NPC provided insights into underlying architectural principles, mechanisms of transport across the nuclear membrane, functional regulation, and assembly/disassembly processes and broadened our understanding of the evolutionary origins of NPCs (Akey et al 2022;Petrovic et al 2022;Bley et al 2022;Zimmerli et al 2021;Allegretti et al 2020;Mosalaganti et al 2018).…”

DGKα, Bridging Membrane Shape Changes with Specific Molecular Species of DAG/PA: Implications in Cancer and Immunosurveillance

“…Each nuclear pore is composed of 500 to 1000 copies of nucleoporin protein as well as PA that accumulate at stalled nuclear pore complexes [ 115 ]. Recently, the architecture of the nuclear pore has been described at high resolution [ 116 , 117 ]. In T cells and cancer cells, serum starvation led to the specific production of saturated and monounsaturated 16 carbon atoms PA species by DGKα, suggesting that these molecular species of PA might have been produced in the nucleus in non-proliferative conditions [ 19 , 20 , 21 ].…”