Architecture of the nuclear pore complex symmetric core

Daniel H. Lin

Tobias Stuwe

Sandra Schilbach

Emily J. Rundlet

Thibaud Perriches

George Mobbs

Yanbin Fan

Karsten Thierbach

Ferdinand M. Huber

Leslie N. Collins

Andrew M. Davenport

Young E. Jeon

, and

André Hoelz

California Institute of Technology, Division of Chemistry and Chemical Engineering, 1200 East

California Boulevard, Pasadena, CA, 91125, USA

Abstract

Introduction—

The nuclear pore complex (NPC) is the primary gateway for transport of

macromolecules between the nucleus and cytoplasm, serving as both a critical mediator and

regulator of gene expression. NPCs are enormous ~120 MDa macromolecular machines embedded

in the nuclear envelope, each containing ~1000 protein subunits, termed nucleoporins. Despite

substantial progress in visualizing the overall shape of the NPC by cryoelectron tomography and

in determining atomic resolution crystal structures of nucleoporins, the molecular architecture of

the assembled NPC remains poorly understood, hindering the design of mechanistic studies that

could investigate its many roles in cell biology.

Rationale—

Existing cryoelectron tomographic reconstructions of the NPC remain too low in

resolution to allow for de novo structure determination of the NPC or unbiased docking of

nucleoporin fragment crystal structures. We sought to bridge this resolution gap by first defining

the interaction network of the NPC, focusing on the evolutionarily conserved symmetric core. We

developed protocols to reconstitute NPC protomers from purified, recombinant proteins, which

enabled the generation of a high-resolution biochemical interaction map of the NPC symmetric

core. We next determined high-resolution crystal structures of key nucleoporin interactions,

providing spatial restraints for their relative orientation. Lastly, by superposing crystal structures

that overlapped in sequence, we generated accurate full-length structures of the large scaffold

nucleoporins. Supported by this biochemical data, we used sequential, unbiased searches to place

the nucleoporin crystal structures into a previously determined cryoelectron tomographic

reconstruction of the intact human NPC, thus generating a composite structure of the entire NPC

symmetric core.

Results—

Our analysis revealed that the inner and outer rings of the NPC utilize disparate

mechanisms of interaction. While the structured coat nucleoporins of the outer ring form extensive

surface contacts, the scaffold proteins of the inner ring are bridged by flexible sequences in linker

Correspondence: hoelz@caltech.edu (A.H.).

these authors contributed equally to this work

Supplementary Materials:

Materials and Methods

Figs. S1–S59

Tables S1–S10

References (

50–67

)

The authors declare no financial conflicts of interest.

HHS Public Access

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Published in final edited form as:

Science

. 2016 April 15; 352(6283): aaf1015. doi:10.1126/science.aaf1015.

Author Manuscript

nucleoporins. Our composite structure revealed a defined spoke architecture with limited cross-

spoke interactions. Most nucleoporins are present in 32 copies, with notable exceptions of Nup170

and Nup188. Lastly, we observed the arrangement of the channel nucleoporins, which orient their

N-termini into two sixteen-membered rings, ensuring that their N-terminal FG repeats project

evenly into the central transport channel.

Conclusion—

Our composite structure of the NPC symmetric core can be used as a platform for

the rational design of experiments to probe NPC structure and function. Each nucleoporin

occupies multiple distinct biochemical environments, explaining how such a large macromolecular

complex can be assembled from a relatively small number of unique genes. Our integrated,

bottom-up approach provides a paradigm for the biochemical and structural characterization of

similarly large biological mega-assemblies.

Graphical Abstract

Composite structure of the nuclear pore complex symmetric core

. The composite structure of

the nuclear pore complex symmetric core generated by sequential, unbiased docking of

nucleoporin and nucleoporin complex crystal structures into the cryoET reconstruction of the

intact human NPC, viewed from above the cytoplasmic face. Nucleoporin structures are shown as

colored cartoons and the nuclear envelope density is shown as a gray surface.

Summary

An integrated analysis of the symmetric core of the nuclear pore complex established its molecular

architecture.

Introduction

The nuclear pore complex (NPC) is a massive molecular transport channel embedded in the

nuclear envelope (

). In addition to its role as the sole mediator of bidirectional

nucleocytoplasmic transport, the NPC is also involved in diverse cellular processes including

transcription, mRNA maturation, and genome organization (

). Despite their tremendous

size (~120 MDa), NPCs are only composed of 34 different proteins (nucleoporins or nups),

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which assemble into eightfold-symmetric, ~1000-Å diameter pores that fuse the inner and

outer nuclear membranes (Fig. 1, A and B) (

). Given their central role in cell biology,

nucleoporins have been linked to a wide range of human diseases including viral infection,

cancer, and neurodegenerative disease (

–

). However, the structure of the NPC and the

mechanisms by which it influences cellular processes remain enigmatic.

Most nucleoporins are symmetrically distributed in the NPC, forming a symmetric core that

is decorated by asymmetric nucleoporins on the nuclear and cytoplasmic faces (Fig. 1, A and

B). Many nucleoporins contain disordered repetitive sequences enriched in phenylalanine

and glycine residues called FG repeats, which collectively form a central diffusion barrier

that prevents passive diffusion of macromolecules with masses greater than ~40 kDa (Fig. 1,

A and B). Larger macromolecules can only traffic through the NPC with the assistance of

specialized karyopherin transport factors (

Despite extensive efforts, the protein-protein interaction network within the NPC remains

incompletely characterized, presenting a fundamental limitation to our understanding of

NPC architecture. Co-purification and mass spectrometry approaches have revealed some of

the strongest interactions, but provide only general spatial restraints due to their limited

resolution. The most well-characterized nucleoporin interactions are those within the coat

nucleoporin complex (CNC), which comprises approximately half the mass of the

symmetric core and contains Nup120, Nup85, Sec13, Nup145C, Nup84, Nup133, and, in

some species, Seh1, Nup37, Nup43, and ELYS (

). Structural and biochemical analyses of

CNCs from multiple species reveal a highly conserved architecture in which

-helical

domains form extensive interaction surfaces to assemble a large, Y-shaped complex (

–

Recent advances have dramatically increased the resolution of cryoelectron tomographic

(cryoET) reconstructions of intact NPCs, facilitating the unbiased placement of 32 copies of

a ~400 kDa crystal structure of the yeast CNC into the intact human NPC (

). The

CNCs are arranged in pairs of concentric, eight-membered rings on both the nuclear and

cytoplasmic faces of the NPC, accounting for the majority of the observed protein density in

the outer rings of the NPC (

In contrast to our understanding of the organization of the CNC in the intact NPC, relatively

little is known about the molecular architecture of the inner ring that lines the central

channel. The disordered N-terminal FG repeats of the channel nucleoporins Nup49, Nup57,

and Nsp1 project into the central channel while their structured coiled-coil domains form a

stable complex, termed the channel nucleoporin hetero-trimer (CNT) (

). The CNT,

Nic96, Nup192, and Nup145N collectively form a stable subcomplex called the inner ring

complex (IRC) (

). The remaining components of the symmetric core, Nup170 and

Nup53, are thought to mediate interactions with the nuclear envelope, but the details of the

interaction network that assembles these proteins and links them to the CNCs remains

poorly defined (

Crystal structures of many nucleoporin fragments from the symmetric NPC core have been

determined, including the N-terminal domains (NTDs) of Nup192, Nup188, and Nup157;

the C-terminal domain (CTD) of Nup170; the C-terminal tail domains (TAIL) of Nup192

and Nup188; the

-helical solenoid of Nic96; and the CNT bound to Nic96, CNT•Nic96

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(

–

). However, structures of full-length Nup192, Nup188, and Nup170 have

remained elusive. Accurate placement of the existing structures into the intact NPC is

limited by both their relatively small size and the resolution of available cryoET

reconstructions. We have used an integrated approach to obtain complete atomic structures

and accurate, high-resolution biochemical restraints for determining the near-atomic

architecture of the NPC symmetric core.

A significant barrier preventing complete biochemical characterization of the NPC has been

the difficulty of purifying significant quantities of full-length nucleoporins from a single

species. To overcome this hurdle, we developed expression and purification protocols for all

symmetric core nucleoporins from the thermophilic fungus

Chaetomium thermophilum

which exhibit superior biochemical stability (

). By reconstituting NPC symmetric core

protomers from purified proteins, we found that the interactions between flexible linker

sequences and large scaffold nucleoporins drove NPC assembly. We generated a high-

resolution biochemical map for these interactions and determined a series of crystal

structures that revealed the structural basis for flexible linker sequence recognition by the

large scaffold nucleoporins. These crystal structures enabled the construction of complete

atomic structures for the ordered scaffolds of Nup170, Nup192, and Nic96. Using our

biochemical restraints for validation, we performed unbiased searches to dock the atomic

structures into a cryoET reconstruction of the intact human NPC with high confidence (

thus determining a composite structure of the NPC symmetric core.

Reconstitution of NPC symmetric core protomers

We first directed our efforts towards reconstituting a soluble protomer that could recapitulate

the interaction network within the assembled NPC using nucleoporins from the thermophilic

fungus

C. thermophilum

. We used full-length proteins when possible, but FG repeats,

disordered N- or C-terminal regions, or other sequences that prevented soluble protein

expression were omitted (Fig. 1B; fig. S1; table S1 and S2). We first reconstituted a hetero-

hexameric core CNC containing Nup120, Nup37, ELYS, Nup85, Sec13, and Nup145C, a

complex analogous to the yeast CNC we previously crystallized (fig. S2, A and B) (

). This

CNC hetero-hexamer was assembled with Nup84 and Nup133 to form a hetero-octameric

CNC (fig. S2C). Due to the poor solubility of the intact hetero-octameric CNC, we focused

our analysis on its hetero-hexameric core. Similarly, we extended our previous reconstitution

of an IRC containing Nup192, Nic96, Nup145N, and the CNT by also incorporating Nup53

(fig. S3A and table S3) (

). We found that Nup188, which is evolutionarily related to

Nup192, failed to incorporate into the IRC (fig. S3B). Rather, an analogous Nup188

complex formed in the absence of Nup192 (Fig. 1F). By preincubating the core CNC-

hexamer with either the IRC or the Nup188 complex, we reconstituted two distinct 13-

protein complexes representative of NPC symmetric core protomers (Fig. 1, C, D, and F and

fig. S4, A and C).

With the ability to reconstitute NPC protomers, we sought to identify the interactions that

linked the IRC or the analogous Nup188 complex to the CNC. Nup145N and Nup145C are

components of the IRC/Nup188 complex and CNC, respectively, and originate from the

same polypeptide chain after post-translational cleavage mediated by the Nup145N

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autoproteolytic domain (APD) located in the middle of the Nup145 pre-cursor polypeptide

(Fig. 1B) (

). Nup145C is composed of a U-bend

-helical solenoid that is a core

component of the CNC and a disordered, ~300-residue N-terminal extension (NTE) (

Previous studies indicate that Nup145N

APD

can still bind the first six N-terminal residues of

Nup145C after cleavage, thus offering a possible mechanism for linking the IRC to the CNC

(

). Indeed, the complexes did not interact in the absence of Nup145N (Fig. 1, E and G and

fig. S4, B and D). Moreover, Nup145N

APD

alone could be incorporated into the CNC (fig.

S5). These results indicated that the IRC and CNC were flexibly attached via the long,

intrinsically disordered sequences of Nup145N and Nup145C. Nup145N

APD

also binds to

the

-propeller domain of the cytoplasmic filament nucleoporin Nup82 (

). However, since

this interaction was outcompeted by CNC binding (fig. S6), another interaction must play a

role in retaining the cytoplasmic filaments at the cytoplasmic face of the NPC.

Symmetric core assembly is driven by flexible linkers

We next performed a systematic analysis of the molecular interaction network within the

IRC and Nup188 complex, extending our previous work and additionally including Nup170

in our analysis (

). These complexes contain large scaffold domains in Nic96, Nup170,

Nup188, and Nup192 as well as long, intrinsically disordered sequences in the linker

nucleoporins Nup53 and Nup145N (Fig. 1B). While the CNC is primarily held together by

extensive interfaces between large

-helical solenoid domains (

), recent studies hint

that the architectural principles of the remaining symmetric core nucleoporins are different

(

). For example, the scaffold nucleoporin Nic96 possesses a largely unstructured

NTE containing two short helical regions, Nic96

and Nic96

, that are essential for IRC

assembly: Nic96

recruits the CNT to the NPC while Nic96

binds Nup192 or Nup188

(

). To determine whether the structured domains of the scaffold nucleoporins interacted

with each other to drive symmetric core assembly, we tested whether Nic96

SOL

, Nup170,

Nup192, and Nup188 could form a complex, but observed no interaction (Fig. 2A and fig.

S7A). Instead, complex formation was achieved only in the presence of the linker

nucleoporins Nup53 and Nup145N (Fig. 2, B and C and fig. S7, B and C). Thus together

with Nic96

NTE

, the flexible linker nucleoporins Nup53 and Nup145N are the primary

driving force of IRC/Nup188 complex assembly (

To further analyze the interaction network between the scaffold and linker nucleoporins, we

tested which scaffolds interacted with Nup53 or Nup145N to form hetero-dimers, focusing

first on the interactions within the IRC and with Nup170. Nup53 formed robust complexes

with Nup192, Nic96

SOL

, and Nup170 (Fig. 2D and fig. S8). We previously reported that

Nup145N interacts weakly with Nic96

SOL

(

), and here we found that Nup145N also binds

to Nup192 and Nup170 (Fig. 2E and fig. S9). All of these scaffold-linker interactions are

compatible, as demonstrated by the formation of hetero-trimeric complexes (Fig. 2, D and E

and figs. S10 and S11). Nup192 and Nup170 can also bind to both linker nucleoporins

simultaneously, indicating that the binding sites on the scaffolds are distinct (fig. S12, A and

B). Indeed, we were able to reconstitute a stoichiometric hetero-tetramer composed of

Nup192, Nup170, Nup53, and Nup145N (fig. S12C).

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To improve the biochemical resolution of our interaction map, we next identified minimal

sequence fragments of Nup53 and Nup145N sufficient for scaffold recognition. We

previously mapped an interaction between a Nup53 fragment, encompassing residues 31–67

(Nup53

), with Nup192

NTD

(Fig. 2F and fig. S13) (

). Here, we found an adjacent

fragment containing residues 69–90 (Nup53

) that was recognized by Nic96

SOL

(Fig. 2F

and fig. S14, A and B). A Nup53 fragment including both of these binding sites (residues 1–

90) was sufficient to link the two scaffolds into a hetero-trimeric complex (fig. S14C). We

also identified a C-terminal Nup53 fragment containing residues 329–361 (Nup53

) that

interacted specifically with Nup170

NTD

(Fig. 2F and fig. S15). Conversely, the association

between Nup170 and Nup145N mapped to Nup145N residues 729–750 (Nup145N

) and

Nup170

CTD

(Fig. 2F and fig. S16). Nup192 recognized a fragment of Nup145N

encompassing residues 606–683 (Fig. 2F and fig. S17, A and B). Nup192

NTD

was sufficient

for Nup145N binding (fig. S17C), but we also detected a weak interaction with Nup192

CTD

(fig. S17, D and E), suggesting that binding sites for Nup145N were distributed throughout

Nup192. These minimal sequence fragments were specific for their binding partners (fig.

S18).

While preincubation of the two linker nucleoporins with Nup192, Nup170, and Nic96

SOL

produced a robust pentameric complex, the analogous preincubation with Nup188 in place

of Nup192 produced a mixture of species (Fig. 2, B and C and fig. S7, B and C). To

understand this difference in behavior, we repeated the above analysis with Nup188 and

identified a robust interaction with Nup145N whereas Nup53 binding was barely detectible

(Fig. 2, D and E and figs. S8C and S9B). However, in contrast to our above results, Nup188

did not strongly bind Nup145N in the presence of Nup192 or Nup170 (Fig. 2, D and E and

fig. S11). Similar to Nup192

NTD

, Nup188

NTD

was sufficient for Nup145N binding (fig. S19,

A and B). However, the minimal Nup192-binding fragment of Nup145N was not sufficient

for Nup188 binding (fig. S19, D and E). Instead, we only detected robust complex formation

with a much longer fragment encompassing both the Nup192 and Nup170 binding sites

(residues 606–750), explaining the exclusivity of their interactions (Fig. 2F and fig. S19C).

We found a similar architecture for the Nup192 and Nup188 binding sites in Nic96

However, the Nup192 minimal binding fragment (residues 286–301) again was insufficient

for Nup188 binding (fig. S20, A to C), which instead required a larger fragment (residues

274–301) (Fig. 2F and fig. S20D). Consistent with these findings, several mutations in the

N-terminal region of Nic96

ablated Nup188 binding but had no effect on Nup192 binding

(fig. S20, E to G) (

). Thus, Nup192 and Nup188 bound competitively to directly

overlapping sequences in Nic96 and Nup145N, establishing the existence of a distinct

Nup188 complex with an architecture analogous to the IRC.

In summary, we found that interactions between the large, ordered scaffold nucleoporins and

flexible interaction motifs in Nup53, Nup145N, and Nic96

NTE

were the dominant driving

force for assembly of the NPC symmetric core outside of the CNCs. We built a biochemical

map of these interactions by identifying minimal interaction motifs, revealing that the

binding sites were spatially distributed throughout the scaffold nucleoporins, but that many

of the binding sites on the linker nucleoporins were adjacent or overlapping in sequence

(Fig. 2F). In doing so, we identified the exclusive interactions that provide a molecular basis

for the formation of two distinct complexes, the Nup192-harboring IRC and an analogous

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Nup188 complex. As existing crystal structures have not captured interactions between

scaffold and linker nucleoporins, we used these results to identify important structural

targets for determining the structural basis for this mode of interaction.

Atomic architecture of the Nup170 interaction network

We determined a crystal structure of the Nup170

NTD

•Nup53

complex at 2.1 Å resolution

(Fig. 3, A and B and tables S4 and S5). In order to obtain high-resolution diffraction, we

deleted residues 293–305 from the 3D4A loop of Nup170

NTD

(fig. S21). Nup170

NTD

was

composed of a seven-bladed

-propeller and a C-terminal

-helical domain (Fig. 3B). An N-

terminal

-helix packed against the C-terminal

-helical domain and was followed by three

-strands that formed a triple Velcro-closure against the

-propeller (Fig. 3B). Nup53

adopted an extended conformation and bound atypically to the side of the

-propeller, rather

than the top, at blades 1 and 2 (Fig. 3B). The crystallized Nup53 fragment contained

residues 329–361, but clear density was only observed for residues 342–355. Blade 2 of the

Nup170

-propeller deviated substantially from a canonical

-propeller blade to generate

two hydrophobic pockets that accommodated Nup53 residues L346, L347, L353, and L354

(Fig. 3C). We identified several mutations in Nup170 and Nup53 that could disrupt their

interaction (Fig. 3, D and E and fig. S22, A and B). Notably, we observed a complete loss of

binding with mutations to Nup170 residues that are evolutionary conserved, F199, I203, and

Y235, suggesting that the binding interface is evolutionarily conserved (Fig. 3, D and E; fig.

S21; fig. S22, A and B).

Nup53 is anchored to the nuclear envelope by its C-terminal amphipathic helix, either

directly or through an interaction with NDC1 (

). Nup170 bound to Nup53

, which is

directly adjacent to this C-terminal helix, prompting us to look for features in Nup170 that

could also contribute to nuclear envelope binding. We identified two motifs next to the C-

terminus of Nup53

that would be juxtaposed with the nuclear envelope. The first was a

WF motif composed of solvent exposed, evolutionarily conserved tryptophan and

phenylalanine residues in the 3CD loop (Fig. 3B and fig. S22C). As tryptophan residues are

enriched at membrane interfaces, the WF motif may reinforce membrane binding (

). The

second motif, residing in the 3D4A loop we deleted for crystallization, is predicted to form

an amphipathic helix with a striking, evolutionarily conserved absence of charged residues, a

feature characteristic of amphipathic lipid packing sensing (ALPS) motifs, which are also

present in Nup120 and Nup133 (fig. S22, C and D) (

). The Nup170 ALPS motif

contained a universally conserved proline residue on the polar face of the helix, a feature

reminiscent of antimicrobial membrane destabilizing peptides (fig. S22D) (

). We propose

that these additional features on Nup170 act synergistically with Nup53 binding to the

nuclear envelope to help maintain the extreme membrane curvature in nuclear pores.

We next determined a crystal structure of the Nup170

CTD

•Nup145N

complex at 3.5 Å

resolution, using a 2.1 Å-resolution structure of

apo

Nup170

CTD

as a search model (Fig. 3, F

and G and tables S4 and S6). Nup170

CTD

formed an elongated

-helical solenoid containing

two stacks of irregular helical pairs, arranged in a zig-zag fashion (Fig. 3G). The two stacks

shared a long helix (

31) that capped the first stack and initiated the second stack.

Nup145N

bound to a pair of deep hydrophobic pockets formed on either side of the first

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two helices of the second helical stack, inserting residues L733 and I735 into the first pocket

and L743 and F744 into the second pocket (Fig. 3, G and H). Mutation of any of these

residues completely abolished binding to Nup170 (Fig. 3, I and J and fig. S23B). Similarly,

mutation of the residues that formed the hydrophobic pockets (F1171, F1154, I1131, and

Y1157) strongly affected binding (Fig. 3, I and J and fig. S23A). The hydrophobic nature of

both binding pockets is retained throughout eukaryotes (fig. S21), suggesting the

evolutionary conservation of this interaction. Only minimal rearrangements of the binding

pocket occur upon Nup145N binding (fig. S23C).

The Nup145N sequence that binds to Nup170 is also highly conserved throughout

eukaryotes, and the homologous residues critical for Nup170 binding are conserved in

humans (fig. S24A). During mitosis, extensive phosphorylation of

Nup98, the human

homologue of Nup145N, leads to NPC and nuclear envelope disassembly (

). The most

abundant mitotic phosphorylation sites in

Nup98 are at residues S608 and S612 (S741 and

S745 in

C. thermophilum

), which flank L610 and F611 (L743 and F744 in

thermophilum

), residues we found to be critical for Nup170 binding (fig. S24, A and B)

(

). To test the possibility that the interaction between Nup170 and Nup145N could be

regulated by phosphorylation, we reconstituted a

Nup155•

Nup98 hetero-dimer

homologous to our crystallized complex. We observed a robust interaction between

Nup155

CTD

and the corresponding minimal

Nup98 fragment, which was partially

disrupted by a phosphomimetic mutation (S608E/S612E) (fig. S24C). As revealed by the

Nup170

CTD

•Nup145N

structure, S612 (S745 in

C. thermophilum

) formed a hydrogen

bond with N609 (D743 in

thermophilum

). Phosphorylation would therefore destabilize

the conformation required to insert the critical hydrophobic residues into the binding pocket

in Nup170 (fig. S24B). Disruption of this hydrogen bond by mutagenesis also completely

abolished binding (Fig. 3J and fig. S23B). Thus, the Nup170-Nup145N interaction is not

only highly conserved, but its disruption is also a key step in mitotic NPC disassembly in

humans.

Our structures of Nup170

NTD

and Nup170

CTD

did not overlap in sequence, preventing

accurate modeling of the full-length protein. Therefore, we crystallized a larger fragment of

Nup170 containing the

-helical solenoids of both domains (Nup170

SOL

, residues 575–

1402), and determined the crystal structure at 4.0 Å resolution (fig. S25A; tables S4 and S5;

movie 1). While there was no conformational variability observed for the helices present in

Nup170

NTD

, the Nup170

CTD

solenoid exhibited an ~20° movement resulting from a minor

rearrangement of helix

27 (fig. S25A). We observed a similar conformational variability in

the Nup170

CTD

•Nup145N

complex structure, where all four molecules in the asymmetric

unit adopted different conformations (fig. S25B). With the structure of Nup170

SOL

as a

template, we superposed the structures of Nup170

NTD

and Nup170

CTD

, obtaining a total of

eight different conformations for full-length Nup170 (figs. S25B and S26 and movie 2).

Molecular basis for recognition of Nup53 by Nic96

We determined crystal structures of

apo

Nic96

SOL

and a Nic96

SOL

•Nup53

complex at 3.3

and 2.65 Å resolution, respectively (Fig. 4, A and B; fig. S27; tables S4 and S7; Movie 3).

Nic96

SOL

formed a rod-shaped molecule consisting of a U-bend

-helical solenoid with the

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N-terminus situated in the middle of the rod. Although residues 31–84 of Nup53 were

included in the crystallization construct, only residues 67–84 were visible in the electron

density. Residues 67–84 of Nup53 formed an amphipathic helix that buried its hydrophobic

face into a hydrophobic groove formed by helices

14,

15, and

16 near the U-bend end

of the Nic96 solenoid (Fig. 4, B and C). Consistent with our crystal structure, we found that

mutations to the hydrophobic residues in this pocket disrupted binding (figs. S28 and S29).

Similar to the interaction between Nup170 and Nup145N, we observed minimal

conformational rearrangements upon Nup53 binding (Fig. 4D).

Structure of Nup192

Nup192, the largest symmetric core nucleoporin, forms a question-mark shape at low

resolution (

). Crystal structures exist for Nup192

NTD

(residues 1–958) and Nup192

TAIL

(residues 1397–1756), but an atomic structure of the entire molecule has remained elusive

(

). To determine the complete atomic structure of Nup192, we obtained crystals of

an engineered Nup192 truncation mutant, Nup192

ΔHEAD

, from which we deleted the N-

terminal HEAD domain (residues 1–152) and replaced a loop encompassing residues 167–

184 with a short glycine-serine linker. We determined the crystal structure of Nup192

ΔHEAD

at 3.2 Å resolution (Fig. 4,E and F; fig. S30; tables S4 and S7).

The N-terminal portion of the previously unresolved middle domain of Nup192

(Nup192

MID

) contained three additional ARM repeats (

46-

53, residues 959–1154) that

continued the superhelical solenoid we previously observed in Nup192

NTD

(Fig. 4F) (

Similarly, the C-terminal portion of Nup192

MID

contained a HEAT repeat (

60-

61,

residues 1330–1376) that extended the Nup192

TAIL

solenoid such that the entire protein

formed a continuous HEAT/ARM repeat solenoid (Fig. 4F) (

). However, we observed an

unusual insertion (residues 1155–1329) between the ARM repeats and HEAT repeat in

Nup192

MID

containing a ~50-residue helix,

58, that reached ~75 Å from the beginning of

Nup192

TAIL

to the C-terminus of Nup192

NTD

(Fig. 4F). This insertion, which we termed the

Tower helix, buried several hydrophobic residues against the bottom of Nup192

NTD

inducing minor rearrangements that facilitate packing of the Tower helix. While the Tower

helix has only a moderate signature in secondary structure predictions, the evolutionarily-

related Nup188 is also predicted to contain a similarly long, ~40 residue helix at the same

location. However, previous models of either full-length Nup188 or Nup192 never

anticipated the existence of the Tower helix, highlighting the importance of experimentally

determining atomic resolution structures (

Taking advantage of the extensive overlap between our crystal structure of Nup192

ΔHEAD

with the existing structures of Nup192

NTD

and Nup192

TAIL

, we generated the structure of

full-length Nup192 by superposition (Fig. 4G and fig S31; Movie 4). Inspection of the full-

length protein revealed that the first loop in the HEAD domain between

1 and

2 was

close enough to contact loops in the MID domain, predominantly with polar and charged

residues (Fig. 4G and fig. S31). The binding sites on Nup192 for Nup53 and Nic96, which

we previously identified via mutagenesis, were located at the top and bottom of the

molecule, respectively, ~140 Å apart from each other (fig. S32) (

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Architecture of the NPC symmetric core

Recent advances in cryoET have produced rapidly improving reconstructions of intact

NPCs, with the most recent reconstructions reporting average resolutions up to ~20 Å in the

best resolved regions (

). We previously docked 32 copies of the yeast CNC into a

~34 Å reconstruction of the intact human NPC, taking advantage of the distinctive shape and

large size of the complex (

). With the addition of the Nic96

SOL

•Nup53

structure and the

full-length, superposition-generated structures of Nup192 and

Nup170•Nup53

•Nup145N

reported here, as well as our recently reported crystal

structure of the CNT•Nic96

complex, accurate structures were available for essentially the

entire ordered mass of the NPC symmetric core (

). The domain architectures of the

symmetric core nucleoporins are highly conserved from fungi to humans (fig. S33). We

successfully located these structures in the recently reported ~23 Å reconstruction of the

human NPC, with the arrangement of nucleoporins validated by our biochemical restraints

(

). We utilized an incremental approach to confidently place the crystal structures, starting

with the largest structures possessing the most distinctive shapes, and iteratively removing

the occupied density to search for subsequent structures (fig. S34). As the cryoET map

possesses eightfold rotational symmetry, each unique solution defined the location and

orientation of eight copies of each molecule. We first tested our approach with the yeast

CNC crystal structure and found four unique placements with exceptional scores compared

to 50,000 other refined placements, in excellent agreement with our previous results (fig.

S35A) (

). We also readily identified the location and orientation of human

Nup84

CTD

•Nup133

CTD

in unbiased searches (fig. S35B) (

). Based on previously reported

biochemical data, we manually docked the Nup37, Nup43, and Nup133

-propellers and

locally optimized their fit (fig. S35C) (

–

We next performed unbiased searches for Nup170 and Nup192 using a map from which

density corresponding to the CNCs had been removed (fig. S36). As our crystallographic

data indicated significant flexibility in the Nup170 solenoid, we performed searches with the

eight different conformations of Nup170. These searches identified two conformations that

each yielded two distinct top scoring solutions (fig. S36, A and B). Searches with full-length

Nup192 revealed six unique solutions, but because Nup192 and Nup188 could not be

distinguished at this resolution, we assigned two of these as Nup188 using our biochemical

results and previously reported cross-linking data, as detailed in the methods (fig. S36C) (

). After removing the density assigned to Nup170, Nup192, and Nup188, we successfully

located four unique copies of the Nic96

SOL

•Nup53

and CNT•Nic96

complexes (fig.

S37, A and B). Lastly, we inspected the remaining density in the inner ring in an attempt to

locate the ordered domains of Nup53 and Nup145N. We determined crystal structures of

Nup53

RRM

and Nup145N

APD

•Nup145C

at 0.8 Å and 1.3 Å resolution, respectively, but

could not unambiguously place them due to their small size and globular shape (fig. S38 and

tables S4, S8, and S9). We attempted to generate biochemical restraints to dock Nup53

RRM

confidently, but were unable to find any binding partners (fig. S39). However, we did find a

pair of continuous densities that readily accommodated two additional Nup170 molecules in

a third distinct conformation (fig. S40A). These placements were buried in our original

global search, but the conformation of this Nup170 structure still differed slightly from the

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remaining map density, suggesting that our crystal structures did not capture the full

conformational range of Nup170 (fig. S40, B and C).

With the CNC-hexamer, Nup84

CTD

•Nup133

CTD

, Nup133

NTD

, Nup37

NTD

, Nup43,

Nup188

NTD

, Nup188

TAIL

, Nup192, Nup170•Nup53

•Nup145N

, Nic96

SOL

•Nup53

and CNT•Nic96

structures placed, we acquired a composite structure accounting for

nearly all of the density in the NPC symmetric core, corresponding to ~54 MDa of protein

mass or ~320,000 ordered residues (Fig. 5; fig. S41; table S10; Movie 5). In total, the

composite structure contained a stoichiometry of 16 copies of Nup188, 32 copies of the

CNC, Nup192, Nic96, and CNT, and 48 copies of Nup170, Nup53, and Nup145N. Overall

this stoichiometry is in good agreement with a previous study that used mass spectrometry to

measure the relative abundances of nucleoporins in the human NPC (

Spoke architecture

The symmetric core of the NPC consisted of eight spokes related by an eight-fold rotational

axis of symmetry perpendicular to the nuclear envelope (Fig. 5A). Outer rings resided above

the nuclear and cytoplasmic faces of the nuclear envelope, while an inner ring was

embedded in the pore and spanned the nuclear envelope (Fig. 5B). When viewed from the

cytoplasm, the nucleoporins formed distinct cylinders, with the CNTs lining the transport

channel, surrounded by successive cylinders formed by Nup192, Nic96, Nup170, and the

CNCs (Fig. 5A). Nic96

NTE

and the linker nucleoporins Nup53 and Nup145N spanned these

cylinders. Only

rotational symmetry was applied to generate the cryoET reconstruction,

yet we observed an additional two-fold axis of symmetry in our composite structure relating

the nuclear and cytoplasmic sides within each spoke (Fig. 6, A to D) (

Each inner ring spoke contained four copies of the IRC (Fig. 6B and Movie 6). Our results

provided spatial restraints for Nic96

and Nic96

, allowing us to trace the path of

Nic96

NTE

, which emerges from the middle of Nic96

SOL

, to its binding sites on Nup192 and

the CNT. We refer to these four distinct IRCs as nuclear peripheral, nuclear equatorial,

cytoplasmic peripheral, and cytoplasmic equatorial IRCs in the following text (Fig. 6B). The

nuclear and cytoplasmic equatorial IRCs were related to each other directly by the two-fold

rotational axis of symmetry, as were the nuclear and cytoplasmic peripheral IRCs.

Unexpectedly, the subunits in the equatorial and peripheral IRCs were in approximately the

same relative orientation, which was readily apparent upon superposition (Fig. 6E). Because

the subunits were placed independently, this surprising symmetry was an emergent property

of the composite structure. The docking reveals that the CNT and Nup192 are in close

proximity, suggesting that additional weaker interactions orient the CNTs in the fully

assembled NPC. We observed additional knobs of density adjacent to the Nup57

domains of each CNT, which were unexplained by our composite structure (fig. S37D).

When we superposed structures of CNT fragments from

Xenopus laevis

onto our docked

CNT molecules (

), we found that the metazoan-specific ferredoxin-like domain of Nup57

accounted for these extra knobs of density (fig. S37, C and D), further validating our

composite structure.

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The mechanism by which FG repeats in the central transport channel form a diffusion barrier

and facilitate transport remains controversial, partly because the stoichiometry and

orientation of the FG repeats remain unknown. Our composite structure revealed a total of

32 CNTs in the inner ring, which would project 96 distinct polypeptide chains in clusters of

three into the central channel (Fig. 6F). The orientation of the CNTs suggested that the FG

repeats would emanate circumferentially towards the adjacent spoke rather than pointing

radially towards the center of the channel. Unexpectedly, the N-termini of the peripheral and

equatorial CNTs were evenly spaced and roughly planar such that they approximately

formed two 16-membered rings (Fig. 6F).

In the outer rings, each spoke contained two CNCs on either face, which we refer to as

proximal and distal CNCs based on their distance from the inner ring. The orientations of

Nup133 relative to the CNC core differed slightly between the distal and proximal CNCs,

but yielded the same overall architecture, as each pair of distal and proximal CNCs formed

an arch over the nuclear envelope (fig. S42). The outer rings also contained a Nup188

molecule on either face (fig. S43). The majority of the CNC components were ~100 Å above

the membrane, and the only contacts with the membrane were made by the

-propeller

domains of Nup120 and Nup133 through their ALPS motifs (Fig. 6C). Similarly, the IRCs

did not make direct contacts with the membrane, but instead were surrounded by a network

of Nup170 molecules that formed the outermost layer of the inner ring (Fig. 6C). Each spoke

contained three distinct pairs of Nup170 molecules, which we refer to as equatorial,

peripheral, and bridging Nup170 molecules. The equatorial pair occupied alternating

orientations equatorially along the surface of the nuclear envelope (Fig. 6C and fig. S36A).

The resolution of the cryoET reconstruction was high enough for these molecules that the

central holes of the

-propellers were readily visible at higher contour levels (fig. S36A).

The bridging pair of Nup170 molecules bridged the inner ring to the outer CNC rings, via a

contact with Nup120 (Fig. 6C and figs. S36B and S43). The peripheral pair of Nup170

molecules, which had a weaker quality of fit and was identified only after placing all other

symmetric core components, contacted both the equatorial and bridging Nup170 molecules

(Fig. 6C and fig. S40). The equatorial Nic96 molecules contacted multiple Nup170

molecules, effectively bridging the nuclear and cytoplasmic networks. Notably, the ALPS

and WF motifs we identified in Nup170

NTD

and the C-terminal amphipathic helix of Nup53

were oriented directly adjacent to the nuclear envelope (Fig. 6C). Thus, Nup170 and Nic96

constitute a membrane coat for the inner ring analogous to the CNCs in the outer ring.

Inter-spoke interactions

We next searched for interfaces mediating interactions between spokes. Several potential

interactions have been identified in previous studies, including one between Nup133

NTE

and

Nup120

NTD

(

). Our composite structure revealed four additional interactions between

CNC components that could link adjacent spokes: (

) between the neighboring proximal

Nup84 and distal Nup85, (

) between the proximal Nup133

-helical solenoid and distal

Nup120

-helical solenoid, (

) between the proximal Nup133

-propeller and proximal

Nup120

-propeller, and (

) between the distal Nup133

-propeller and distal Nup120

propeller (fig. S43, A to C). The space between the proximal and distal CNCs contained

density that readily accommodated a Nup188 molecule. Nup188 recognized a special niche

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generated in the CNC inter-spoke interface, bridging four CNC molecules by potentially

making contacts with: (

) the distal Sec13, (

) the distal Nup85, (

) the proximal Nup43,

and (

) the proximal Nup133 of a neighboring spoke (fig. S43, B and C). The Nic96

binding site in Nup188

TAIL

was not occluded by any additional density (fig. S43B). Due to

the absence of strong density in this region, we cannot determine whether only Nup188 or

entire an Nup188 complex would be anchored to the outer rings at this site. However, any

flexibly tethered components of the NPC, including the remainder of the Nup188 complex,

may not be clearly visible in cryoET reconstructions.

Inspection of the interaction surfaces also provided a molecular explanation for how two

CNC rings are assembled. Nup133 and Nup170 bind to distinct Nup120 molecules via

overlapping interfaces on the proximal and distal Nup120 molecules, respectively,

effectively capping the CNCs on either side (Figs. 43, D and E). These results are in

agreement with a common evolutionary origin for Nup120, Nup133, and Nup170, which all

possess similar domain architectures, contact the nuclear envelope via ALPS motifs, and

interact with each other at inter-spoke interfaces. The U-bend solenoid nucleoporins, Nup85,

Nup145C, Nup84, and Nic96, bridge these inter-spoke interfaces to form a continuous

membrane-bending coat. These results also support the protocoatomer hypothesis of a

common evolutionary origin for vesicle coats and the NPC coat, wherein the extant

nucleoporins derived from an ancient membrane coat containing these protein folds (

We could only identify a single interaction that would analogously link the inner ring

spokes, which would be mediated by an interaction between Nup53

and Nup192 (Fig. 7).

In our composite structure, peripheral Nic96 molecules oriented Nup53

directly adjacent

to the Nup53-binding site on equatorial Nup192 molecules from a neighboring spoke (Fig.

7B). Our biochemical mapping experiments identified adjacent binding sites for Nic96 and

Nup192 on Nup53 (Fig. 2F) and that a fragment containing both these binding sites could

bridge the two nucleoporins (fig. S14C). Thus, binding of Nup53

in trans to a Nup192

molecule from a neighboring spoke would link the inner ring spokes.

An open question regarding nucleocytoplasmic transport has been the mechanism of inner

nuclear membrane (INM) protein transport through the NPC, particularly for INM proteins

with large globular nuclear domains. Peripheral channels on the order of ~100 Å have been

proposed as routes for INM transport (

), but we observed no such channels through the

inner ring in either our composite structure or the cryoET reconstruction (Fig. 5). Given the

dense packing within each spoke, traffic of INM proteins through a spoke would require

significant disruption of NPC structure (Fig. 7A). Rather, the most likely path through the

inner ring would be at the inter-spoke interfaces where Nup53

and Nup192 interact (Fig.

7). The CNCs form an ~100-Å arch above this interface, providing an uninterrupted path to

the inner ring and possibly explaining the previously observed upper limit for the size of

nuclear domains (Fig. 6A) (

). However, the channel at the inner ring was much smaller,

suggesting that rearrangements at the inter-spoke interface may be necessary to traffic large

nuclear domains. Thus, our composite structure of the NPC symmetric core enables rational

design of experiments to further understand the mechanism of INM protein import.

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A flexible linker mediates CNC oligomerization

While reconstituting the CNCs, we noticed that assembly of the CNC-octamer

spontaneously generated a separate solution phase (fig. S44A). Similar phase transitions

were previously seen in other systems with multiple binding valencies, suggesting that the

oil droplet formation we observed resulted from oligomerization of the CNC (

). We

previously identified an interaction between Nup133

NTE

and Nup120 that would mediate

head-to-tail CNC ring formation consistent with the composite structure of the NPC (

Removal of the unstructured Nup133

NTE

completely ablated both oil droplet and complex

formation, suggesting that this flexible interaction serves as a driving force for CNC ring

formation (fig. S44). We also found that Nup170 could be incorporated into this separate

solution phase and that this incorporation was ablated by C-terminal truncation, which was

consistent with the interaction between the proximal Nup120 and bridging Nup170

molecules we observed in our composite structure (fig. S44A).

Conservation of NPC architecture

Our results establish the principles that drive the assembly of nucleoporins in the NPC.

While the outer rings assemble largely via structurally rigid interaction surfaces, inner ring

assembly is primarily driven by flexible linker sequences within Nup53, Nup145N, and

Nic96

NTE

. This dichotomy may reflect the different roles of the respective complexes. The

outer rings provide a structural scaffold for the NPC and given their location above the plane

of the nuclear envelope, their assembly would not be affected dramatically by the dynamic

generation of membrane curvature during fusion of the inner and outer nuclear membranes.

In contrast, the proteins in the inner ring occupy an environment that only exists after

membrane fusion, likely necessitating conformational flexibility over the course of NPC

assembly.

The importance of these flexible interactions in the NPC is highlighted by their evolutionary

conservation despite poor overall sequence conservation in the linker nucleoporins Nup53

and Nup145N. The overall folds of the scaffold proteins are well-conserved in

S. cerevisiae

(fig. S45) and furthermore, point mutations in the binding pockets of

S. cerevisiae

Nic96,

Nup170, and Nup157 also disrupted their interaction with linker sequences (figs. S46 to

S48). A complete understanding of the interaction network in

S. cerevisiae

has been partially

intractable because of the genetic redundancy that arises from several gene duplications

(Nup170/Nup157, Nup53/Nup59, and Nup145N/Nup100/Nup116). We found that the

paralogs mostly retained the ability to form these interactions, but did not detect an

interaction between

Nup188 and any of the Nup145N paralogs (figs. S46 to S50).

Our structural data also highlighted the evolutionary conservation of nucleoporin structure

and their interactions. While crystal structures of the human scaffold nucleoporins have not

been determined, previous comparisons of the fungal CNC with low-resolution

reconstructions of the human CNC suggest a conserved architecture (

). Superposition

of the structures of Nup53

RRM

and Nup145N

APD

with their human homologues also

revealed that their folds were identical (fig. S38) (

). In addition, the mechanism of

interaction between Nup170 and Nup145N is conserved in humans, and we found that

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phosphomimetic mutations weakened the interaction between

Nup98 and

Nup155.

Several other phosphorylation sites have been identified in Nup98, many of which

potentially overlap with scaffold binding sites (

). Therefore, phosphorylation could also

regulate other key interactions, including those that occur between Nup145N and Nup188,

Nup192, and the CNC. We note that Nup53 is similarly phosphorylated in a cell-cycle

dependent manner, and thus phosphorylation of both linker nucleoporins would be an

effective means to disassemble the entire inner ring of the NPC (

Conclusions

Determining the molecular details of nucleocytoplasmic transport has been a longstanding

challenge, at least in part due to an incomplete understanding of the architecture and

biochemistry of the NPC itself. We have used purified, recombinant proteins to

systematically characterize the nucleoporin interaction network and determine atomic

resolution structures of nucleoporin complexes. This approach was crucially complemented

by recent advances in cryoET reconstructions (

). Using the results of our divide-and-

conquer approach, we were able to dock the available crystal structures into a cryoET

reconstruction of the human NPC, yielding a composite structure for the entire NPC

symmetric core. This union of bottom-up and top-down approaches offers a paradigm for

determining the architectures of similarly complex macromolecular assemblies.

Our composite structure differs dramatically from the previously reported computational

models not only in relative and absolute stoichiometry, but also in overall architecture (

These discrepancies highlight the complexities that must be accommodated when attempting

a holistic, computational approach. We observed a remarkable degree of symmetry in the

structure of the NPC, which explains how such a limited vocabulary of proteins can generate

such a large macromolecular structure. Most nucleoporins also occupy multiple, distinct

biochemical environments. Nup170 offers a dramatic example of this property, as

biochemically distinct versions of Nup170 are either buried in the inner ring or are exposed

in the bridge between the inner and outer rings. Similarly, Nup120 utilizes overlapping,

exclusive interfaces to contact Nup170 and Nup133. Due to this diversification of

nucleoporin function, the NPC can be encoded by a relatively small number of genes. The

gene duplications of nucleoporins in

S. cerevisiae

may reflect the gradual separation of these

distinct functions into several genes. Nup170 appears to also adopt different conformations

in each of its distinct biochemical environments, which is consistent with the wide

conformational range we observed in crystal structures. It is possible that the different

conformations are the result of different mechanical forces acting on Nup170 at each

position.

Biochemical diversification of proteins within the same protein complex also generates

enormous challenges for computationally modeling the structure of the NPC and similar

complexes, as distance restraints such as crosslinks that are valid in one biochemical

environment may be violated in another. This challenge is exacerbated by the possibility of

flexibly tethered domains or nucleoporins, such as those in the Nup188 complex. Our results

also highlight the confounding effect generated by the flexible linker nucleoporins Nup53

and Nup145N, which occupy binding sites that span the entirety of the inner ring, rather than

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