Architecture of the nuclear pore complex coat

Tobias Stuwe

1,3

Ana R. Correia

1,3

Daniel H. Lin

Marcin Paduch

Vincent T. Lu

Anthony

A. Kossiakoff

, and

André Hoelz

1,*

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

California Boulevard, Pasadena, CA 91125, USA

Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, Illinois

60637, USA

Abstract

The nuclear pore complex (NPC) constitutes the sole gateway for bidirectional nucleocytoplasmic

transport. Despite half a century of structural characterization, the architecture of the NPC remains

unknown. Here, we present the crystal structure of a reconstituted ~400 kDa coat nucleoporin

complex (CNC) from

S. cerevisiae

at a 7.4-Å resolution. The crystal structure revealed a curved Y-

shaped architecture and the molecular details of the coat nucleoporin interactions forming the

central “triskelion” of the Y. A structural comparison of the yeast CNC with an electron

microscopy reconstruction of its human counterpart suggested the evolutionary conservation of the

elucidated architecture. Moreover, thirty-two copies of the CNC crystal structure docked readily

into a cryoelectron tomographic reconstruction of the fully-assembled human NPC, thereby

accounting for ~16 MDa of its mass.

The nuclear pore complex (NPC) is composed of ~34 different proteins, termed

nucleoporins (nups), that assemble in numerous copies to yield a ~120 MDa transport

channel embedded in the nuclear envelope (NE) (

). To facilitate the extensive membrane

curvature generated in each NE pore, NPCs require a membrane-bending coat. The NPC

coat is believed to be formed by an evolutionarily conserved coat nup complex (CNC), the

Nup107/160 complex in humans and the Nup84 complex in

S. cerevisiae

, the latter of which

is composed of Nup120, Sec13, Nup145C, Seh1, Nup85, Nup84, and Nup133 (

We reconstituted a hetero-hexameric CNC containing the yeast nups Nup120, Sec13,

Nup145C, Seh1, Nup85, and the Nup84 N-terminal domain (NTD) (Fig. 1, A and B). Our

reconstituted CNC did not include Nup133 because this nup is conformationally flexible and

loosely associated (

–

). Because the initial crystals of this reconstituted CNC diffracted

poorly, we generated a series of conformation-specific, high-affinity synthetic antibodies

Corresponding author: hoelz@caltech.edu (A.H.).

These authors contributed equally to this work

SUPPLEMENTARY MATERIALS

Materials and Methods

Figures S1–S13

Tables S1–S2

Movies S1–S4

References (

–

)

HHS Public Access

Author manuscript

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. Author manuscript; available in PMC 2016 December 23.

Published in final edited form as:

Science

. 2015 March 06; 347(6226): 1148–1152. doi:10.1126/science.aaa4136.

Author Manuscript

(sABs) and tested them as crystallization chaperones (

). This approach yielded crystals of

the CNC in complex with sAB-57, which allowed us to solve the structure to 7.4 Å by

molecular replacement using high-resolution crystal structures of CNC components and the

sAB scaffold (fig. S1 and S2) (

–

). The inclusion of a second sAB (sAB-87) produced

another crystal form, for which we collected anomalous X-ray diffraction data of Seleno-L-

methionine and heavy metal-labeled crystals to confirm the placement of the CNC

components (fig. S1, S2 and S3). Because the coat nups in both CNC•sAB complexes

adopted the same arrangement, we focused our analysis on the better ordered CNC•sAB-57

structure (fig. S4, S5, and S6).

The CNC adopted a curved Y-shaped structure spanning ~250 Å in length and width,

consistent with previous negative-stain electron microscopy (EM) analyses (Fig. 1C and

movie S1) (

–

). The Seh1•Nup85 pair and Nup120 constituted the upper arms of the Y,

which were connected to the rest of the CNC through a central triskelion.

Sec13•Nup145C•Nup84

NTD

formed the stalk at the bottom of the triskelion and would

attach the tail formed by Nup84

CTD

and Nup133, which were absent in the structure. Both

arms curved out such that the Nup120

-propeller domain was perpendicular to the plane of

the Y. Nup145C organized the CNC through four distinct interaction surfaces contacting

nearly every member of the complex. sAB-57 bound at the Nup145C-Nup85 interface and

formed crystal packing contacts (Fig. 2 and fig. S4).

The C-terminal domains (CTDs) of Nup145C (residues 553–712), Nup85 (residues 545–

744), and Nup120 (residues 729–1037) converged to form the CNC triskelion. While we

observed clear electron density that revealed the connectivity of the three CTDs and their

interactions (Fig. 2 and fig. S2), the sequence register in the triskelion was only approximate

due to the absence of side chain density. Nup120

CTD

was sandwiched between Nup85

CTD

and Nup145C

CTD

and no direct contacts were observed between Nup85

CTD

and

Nup145C

CTD

(Fig. 2, A and B). The interactions between Nup85

CTD

, Nup145C

CTD

, and

Nup120

CTD

were mediated predominantly by their most C-terminal helices. An additional

interaction was made by an N-terminal Nup145C helix bound to a groove in the Nup85

CTD

surface ~60 Å away from the triskelion center, an interaction that was recognized by sAB-57

(Fig. 2C).

Consistent with our structural data, we reconstituted a stoichiometric complex between

Nup120 and Nup85

CTD

as monitored by size-exclusion chromatography interaction

experiments (fig. S7A). Furthermore Nup120 failed to interact with Sec13•Nup145C in the

absence of Nup145C

CTD

(fig. S7, B and C). The interaction between Seh1•Nup85 and

Sec13•Nup145C depended on the presence of an N-terminal Nup145C fragment (residues

75–125) (fig. S7, D and E). Further mapping identified a region of Nup145C (residues 75–

109) that was sufficient for Nup85

CTD

binding (fig. S7F), confirming that this fragment

bridged the two subcomplexes. Nup120

CTD

and Nup85

CTD

were essential for the formation

of the CNC (fig. S8 and S9). These data were consistent with published CNC cross-linking

data of three different species, more so than the models generated by coarse-grained analysis

(

). Lastly, to validate their placement in the structures, we confirmed the

interactions of sAB-57 and sAB-87 with Seh1•Nup85•Nup145C

1-123

and Nup120

NTD

respectively (fig. S10).

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Next, we compared the CNC structure to previously determined EM reconstructions of the

yeast and human CNCs (

). While the EM reconstruction of the yeast CNC recapitulates

its overall shape, significant deviations were apparent (fig. S11 and movie S2). No density is

observed in the EM structure for the U-shaped tip of Nup85. The overall shape of the crystal

structure was also consistent with the human CNC EM reconstruction, which contains the

crystallized evolutionarily conserved core as well as two additional human components,

Nup37 and Nup43 (Fig. 3A and movie S3). Our crystal structure did not account for

additional EM density directly adjacent to the Seh1•Nup85 and Nup120 arms, which

reportedly accommodate Nup43 and Nup37, respectively (Fig. 3A) (

). In the human CNC,

Nup43 appears to bind to the same site as sAB-57 on the Seh1•Nup85 arm of the Y. The

major difference between the CNC crystal structure and both EM reconstructions is the

curvature of the arms of the Y, and thus the orientation of the Nup120

-propeller was

substantially different in the crystal structure (Fig. 3A). The flatness of both EM

reconstructions suggests that these deviations may be a result of EM sample preparation.

Despite this, the degree of similarity between the yeast CNC crystal structure and the human

CNC EM reconstruction suggested substantial evolutionary conservation of the CNC

architecture.

The higher-order arrangement of CNCs in a fully assembled NPC has been debated, with

several models proposed based on various structural, biophysical, or computational

approaches (

). Given the evolutionary conservation of the CNC architecture, we

tested whether our crystal structure could be docked into the ~32-Å resolution tomographic

reconstruction of an intact human NPC (

). Indeed, an unbiased 6-dimensional search

combined with a cross-correlation analysis confidently docked 32 copies of the CNC crystal

structure in the tomographic reconstruction, yielding a model for the NPC coat (Figs. 3B, 4A

and S12). These results agreed with the stoichiometry and approximate localization

previously proposed based on crosslinking mass spectrometry and the docking of the human

CNC EM reconstruction (

). However, the crystal structure fit the tomographic

reconstruction substantially better than the human CNC EM reconstruction (Fig. 3B).

The NPC coat was formed by 32 copies of the CNC arranged in four eight-membered rings

(Fig. 4, A and B and movie S4). The eight CNCs in each ring were oriented horizontally

with their long axis positioned parallel to the surface of the NE in a head-to-tail fashion. On

each side of the NE a pair of inner and outer CNC rings emerged up to ~210 Å (Fig. 4A).

These rings were separated by a ~280 Å gap, yielding a total height of ~700 Å. The

diameters of the outer CNC rings were slightly larger than those of the inner CNC rings,

spanning ~1,200 Å and ~1,050 Å, respectively (Fig. 4B). While the CNCs in both rings were

arranged with the same directionality, each CNC from the outer ring was offset from its mate

in the inner ring by ~120 Å in a clockwise direction (Fig. 4B). Moreover, the tandem CNC

rings on the nuclear and cytoplasmic side of the NE possessed the same handedness and

were related by two-fold rotational symmetry (Fig. 4A and S12D).

The unambiguous placement and orientation of the coat nups and their conserved surfaces

allowed for an investigation into the details of their interactions in the assembled NPC coat.

Each CNC was situated on top of the NE and was oriented such that the plane of the Y was

nearly perpendicular to the membrane, with the Nup120 and Seh1•Nup85 arms pointed at or

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away from the membrane, respectively (Fig. 4A). Only two interfaces appeared to be

responsible for oligomerization of individual CNCs into the NPC coat. The inner and outer

rings only interacted where the top of the triskelion of each inner ring CNC met the bottom

of the Nup84-Nup145C interface of each outer ring CNC (Fig. 4C). Nup120 was oriented

such that its Nup133 binding site was directly adjacent to the density assigned to the N-

terminus of Nup133, consistent with our previous findings that this interaction is responsible

for CNC ring formation and critical for NPC assembly (Fig. 4D) (

). This Nup120

orientation also pointed the apex of its

-propeller directly towards the NE, which was the

only membrane contact that we observed in our model (Fig. 4D). This region of the Nup120

-propeller domain also contains a conserved surface patch on its side (

) that may serve as

a NE anchor point for the entire NPC coat, either through a direct interaction with the

membrane or via a membrane-anchored nup, as previously reported (

The NPC coat architecture is dissimilar to other structurally characterized membrane coats.

Whereas the latter are generated by homotypic vertex elements (

), the NPC coat is

formed by heterotypic interactions of its asymmetric CNC protomers. Given the location of

the CNC rings above and below the NE, other nups likely play a role in generating the

complex curvature of the NE pores. While the placement of the CNCs in the NPC coat did

not directly address the organization of the central transport channel (fig. S13), it accounted

for ~16 MDa of the total mass of the NPC, bridged the resolution gap between low-

resolution EM analyses and high-resolution crystallographic studies, and suggested the

evolutionarily conservation of its architecture.

Supplementary Material

Refer to Web version on PubMed Central for supplementary material.

Acknowledgments

We thank C.J. Bley, W.M. Clemons, A.M. Davenport, O. Dreesen, A. Patke, D.C. Rees, S.O. Shan, P.

Stravropoulos, and K. Thierbach for critical reading of the manuscript, K. Kato and K. Kato for their contributions

at the initial stages of this project, L.N. Collins for technical support, D. King for mass spectrometry analysis, E.

Hurt and P. Loppnau for material, S. Koide for providing the phage display library, S. Gräslund for the pSFV4

vector, P. Afonine for advice regarding structure refinement in PHENIX, and J. Kaiser and the scientific staff of

SSRL Beamline 12-2 and the APS Beamline GM/CA-CAT for their support with X-ray diffraction measurements.

We acknowledge the Gordon and Betty Moore Foundation, the Beckman Institute, and the Sanofi-Aventis

Bioengineering Research Program for their support of the Molecular Observatory at the California Institute of

Technology. The operations at the SSRL and APS are supported by the Department of Energy and the National

Institutes of Health. T.S. was supported by a Postdoctoral Fellowship of the Deutsche Forschungsgemeinschaft.

D.H.L. was supported by a National Institutes of Health Research Service Award (5 T32 GM07616). A.A.K. was

supported by National Institutes of Health Awards (U01 GM094588, U54 GM087519) and the Chicago Biomedical

Consortium. A.H. was supported by Caltech startup funds, the Albert Wyrick V Scholar Award of the V Foundation

for Cancer Research, the 54

Mallinckrodt Scholar Award of the Edward Mallinckrodt, Jr. Foundation, and a

Kimmel Scholar Award of the Sidney Kimmel Foundation for Cancer Research. The coordinates and structure

factors have been deposited with the Protein Data Bank with accession codes 4XMM and 4XMN. The authors

declare no financial conflicts of interest.

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Fig. 1. Overall architecture of the CNC

(

) Domain structures of the yeast coat nups and sAB-57. Black lines indicate the

crystallized fragments. U: unstructured, D: domain invasion motif, VH: heavy chain variable

region, CH: heavy chain constant region, VL: light chain variable region, CL: light chain

constant region. (

) Reconstitution of the yeast CNC•sAB-57, lacking Nup133. Elution

profiles from a Superdex 200 10/300 column are shown for Nup120•Seh1•Nup85 (Trimer

1), Sec13•Nup145C•Nup84

NTD

(Trimer 2), CNC, and CNC•sAB-57 (left). SDS-PAGE gel

of the reconstituted CNC•sAB-57 used for crystallization (right). (

) Cartoon and schematic

representations of the yeast CNC•sAB-57 crystal structure viewed from two sides.

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Fig. 2. Architecture of the CNC triskelion

Cartoon representation of the triskelion formed by Nup120, Nup85 and Nup145C. Insets

(

A–C

) depict magnified views for the interactions between (

) Nup120

CTD

, Nup85

CTD

, and

Nup145C

CTD

(

) Nup120

CTD

, Nup85

CTD

, and N-terminal Nup145C helix; and (

)

Nup145C, Nup85

CTD

, and sAB-57. The density modified electron density map is contoured

at 1.0

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Fig. 3. Comparison of yeast and human CNCs

(

) Fit of the yeast CNC crystal structure into the human CNC negative-stain EM

reconstruction (gray) (

). Arrows indicate density accounted for by the additional human

coat nups Nup37 or Nup43. (

) Comparison of the quality of fit for the yeast CNC crystal

structure and human CNC EM reconstruction (cyan) into the intact human NPC cryoelectron

tomographic reconstruction (gray) (

). Arrows indicate regions where the human CNC EM

reconstruction protrudes from the cryoelectron tomographic reconstruction.

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Fig. 4. Architecture of the NPC coat

(

) 32 copies of the yeast CNC, shown in cartoon representation with a representative

subunit colored as in Fig. 1, docked into the cryoelectron tomographic reconstruction of the

intact human NPC (

), shown as a gray surface. The outer and inner cytoplasmic and nuclear

CNC rings are highlighted in orange, cyan, pink, and blue, respectively. (

) Cartoon

representations of 16 yeast CNC copies from the cytoplasmic side of the NPC coat.

Schematics indicating the positions assigned to Nup84

CTD

and Nup133, which were not

crystallized, are shown. (

) Interface between the inner and outer CNC rings. Two views of

the yeast CNC and its mate from the inner ring are shown. (

) Orientation of the Nup120

propeller relative to neighboring coat nups and the membrane. Portions of two CNCs from

the cytoplasmic outer ring are shown in cartoon representation. Green and cyan shading

indicates the positioning of Nup84

CTD

and Nup133, respectively. The cyan line represents

the N-terminal unstructured segment of Nup133 that binds to Nup120 (

). A schematic

representation of the ring-forming Nup120-Nup133 interaction is shown below.

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