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Architecture of the linker-scaffold in the nuclear pore
Stefan Petrovic
1
,
Dipanjan Samanta
1,#
,
Thibaud Perriches
1,#,†
,
Christopher J. Bley
1
,
Karsten Thierbach
1,‡
,
Bonnie Brown
1
,
Si Nie
1
,
George W. Mobbs
1
,
Taylor A. Stevens
1
,
Xiaoyu Liu
1,§
,
Giovani Pinton Tomaleri
1
,
Lucas Schaus
1
,
André Hoelz
1,*
1
California Institute of Technology, Division of Chemistry and Chemical Engineering, 1200 East
California Boulevard, Pasadena, CA 91125, USA
Abstract
Nuclear pore complexes (NPCs) mediate the nucleocytoplasmic transport of macromolecules.
Although the arrangement of the structured scaffold nucleoporins in the NPC’s symmetric core
had been determined, their cohesion by multivalent unstructured linker nucleoporins 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 ~64MDa 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.
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 ~110MDa human NPC is an ~1,200Å wide and ~750Å tall assembly
of ~1,000 proteins, collectively termed nucleoporins. Because of the NPC’s eight-fold 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
*
Corresponding author. hoelz@caltech.edu (A.H.).
Present address: Care Partners, 146 chemin de l’Etang, 69380 Dommartin, France.
Present address: Odyssey Therapeutics, Inc., Industriepark Höchst, G875, 65926 Frankfurt am Main, Germany.
§
Present address: Department of Microbiology, Immunology & Molecular Genetics, University of California, Los Angeles, 609
Charles E. Young Drive East, Los Angeles, CA 90095, USA.
#
These authors contributed momentously and equally to this work.
Author contributions:
AH conceived and coordinated the study. SP, DS, TP, CJB, KT and AH designed research. SP, DS, TP, CJB,
KT, BB, GPT, and LS performed research. SP, DS, TP, CJB, KT, BB, SN, GWM, TAS, XL, GPT, LS, and AH analyzed data. SP,
DS, TP, CJB, and AH integrated and conceptualized the results. SP, CJB, SN, GWM, and AH wrote and revised the manuscript, with
contributions from all authors.
Competing interests:
The authors declare no financial conflicts of interest.
Supplementary Materials:
Materials and Methods
Supplementary Text
Figs. S1 to S83
Tables S1 to S16
References (
90
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Movies 1 to 5
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. Author manuscript; available in PMC 2023 January 21.
Published in final edited form as:
Science
. 2022 June 10; 376(6598): eabm9798. doi:10.1126/science.abm9798.
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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 ~200Å. 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 is not only 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 via short defined motifs, with flanking regions commonly forming additional
disperse interactions that reinforce the binding. Employing a structure-guided functional analysis
in
S. 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, flexibly inter-connected 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.
CONCLUSIONS—
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 cargoes, and the anchoring of asymmetric nucleoporins.
Petrovic et al.
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