Simple fold composition and modular architecture
of the nuclear pore complex
Damien Devos*, Svetlana Dokudovskaya†, Rosemary Williams†, Frank Alber*, Narayanan Eswar*, Brian T. Chait‡,
Michael P. Rout†§, and Andrej Sali*§
*Departments of Biopharmaceutical Sciences and Pharmaceutical Chemistry and California Institute for Quantitative Biomedical Research, University of
California, Mission Bay QB3, 1700 4th Street, Suite 503B, San Francisco, CA 94143-2552; and Laboratories of†Cellular and Structural Biology and
‡Mass Spectrometry and Gaseous Ion Chemistry, The Rockefeller University, 1230 York Avenue, New York, NY 10021-6399
Edited by Peter Walter, University of California School of Medicine, San Francisco, CA, and approved December 23, 2005 (received for review July 26, 2005)
The nuclear pore complex (NPC) consists of multiple copies of ?30
different proteins [nucleoporins (nups)], forming a channel in the
nuclear envelope that mediates macromolecular transport be-
tween the cytosol and the nucleus. With <5% of the nup residues
currently available in experimentally determined structures, little
is known about the detailed structure of the NPC. Here, we use a
combined computational and biochemical approach to assign folds
for ?95% of the residues in the yeast and vertebrate nups. These
fold assignments suggest an underlying simplicity in the compo-
simplicity in NPC composition is reflected in the presence of only
eight fold types, with the three most frequent folds accounting for
?85% of the residues. The modularity in NPC architecture is
reflected in its hierarchical and symmetrical organization that
partitions the predicted nup folds into three groups: the trans-
membrane group containing transmembrane helices and a cad-
herin fold, the central scaffold group containing ?-propeller and
?-solenoid folds, and the peripheral FG group containing predom-
inantly the FG repeats and the coiled-coil fold. Moreover, similar-
ities between structures in coated vesicles and those in the NPC
support our prior hypothesis for their common evolutionary origin
in a progenitor protocoatomer. The small number of predicted fold
types in the NPC and their internal symmetries suggest that the
bulk of the NPC structure has evolved through extensive motif and
coated vesicle ? protocoatomer ? evolution ? fold assignment
(NE) (1). The NPC is also one of the largest assemblies of defined
structure in the cell, with a size of ?50 MDa in yeast and up to 100
MDa in vertebrates. NPCs are common to all eukaryotes and are
composed of a broadly conserved set of proteins termed nucleo-
porins (nups) (2) that have been fully cataloged for both yeast (3)
and vertebrates (4).
Structural characterization of the whole NPC has proven chal-
lenging, because of its size and flexibility. A consensus low-
resolution map of the NPC has emerged based largely on electron
cryomicroscopy and tomography studies (5–8). The NPC is a ring
of eight identical spokes. Each spoke can be divided into almost
identical cytosolic and nuclear half-spokes that each consist of ?25
different nups (1). At the center of the NPC is an aqueous channel
serving as the conduit for the transport of macromolecules. Mac-
romolecular transport is regulated by the filamentous FG repeat-
containing nups that emanate from the NPC into the nucleoplasm
and cytoplasm. A comparison of the vertebrate and yeast NPCs
reveals that the main features of the complex are conserved (1, 2).
able for only seven nup fragments: ?20 residues of the Nsp1 FxFG
repeat region (9), 38 residues of the C terminus of Nup1 (10), a
of Nup159) (11), the autocatalytic fragment of the vertebrate
Nup98 (12), the equivalent 147-residue NPC-targeting domain at
for the passage of macromolecules across the nuclear envelope
the C terminus of Nup116 (13), and the N-terminal domains of
human Nup133 (14) and yeast Nup159 (15). Together, these
domains represent a mere 5% of the total number of nup amino
acid residues. Moreover, only an additional 5% of the residues can
be related to proteins of known structure via statistically significant
sequence similarity (Results); hence, there is a paucity of high-
resolution structural data on nups.
Despite the central role of the NPC in the cell biology of all
modern eukaryotes, there has been until recently little information
concerning its origin and evolution in protoeukaryotes. To address
this question, it would be helpful to have structural characteriza-
tions of many nups because it is easier to recognize similarities in
structure than in sequence, especially for distantly related proteins.
We recently used bioinformatics tools supported by limited prote-
olysis data to assign folds to domains in seven nups comprising the
Nup84 subcomplex in yeast (16). These assignments allowed us to
propose an evolutionary relationship between the Nup84 subcom-
plex of the NPC and coated vesicles (16), and other relationships
have subsequently been suggested (17). In this work, we extend our
previous analysis and assign folds for domains in all known nups,
resulting in a structural characterization of ?95% of the nup
residues. We discuss the implications of these fold assignments for
the structural organization and evolution of the NPC.
We applied a variety of bioinformatics methods to characterize the
structures of the Saccharomyces cerevisiae nups (Fig. 1 and Table 1)
(3) and their predicted vertebrate homologs (4). We first predicted
secondary structure segments, transmembrane helices (TMHs),
coiled coils, FG repeats, and disordered regions. Second, we
in fold assignments for domains in some nups. Third, we also
assigned folds to as many nup domains as possible using threading
methods. And finally, for a given fold assignment, we applied an
iterative process of sequence–structure alignment, comparative
model building, and model evaluation to assess its accuracy. The
fold assignments were also supported by protease-accessibility
Fold Assignments. The 28 nups were divided into 44 domains as
follows. Using sequence analysis, we detected 12 FG repeats, 5
coiled-coil, and 3 TMH domains. An additional five domains could
be assigned to a fold type by PP?SCAN based on statistically signif-
to assign folds to the remaining 19 domains. The 44 domains
together account for ?95% of all nup residues. Each nup has at
Conflict of interest statement: No conflicts declared.
This paper was submitted directly (Track II) to the PNAS office.
Abbreviations: NPC, nuclear pore complex; NE, nuclear envelope; nup, nucleoporin; TMH,
transmembrane helix; RRM, RNA recognition motif.
§To whom correspondence may be addressed. E-mail: email@example.com or rout@
© 2006 by The National Academy of Sciences of the USA
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least one assigned domain. The only unassigned regions with ?50
N termini of Nup145C, Nup85, Nup133, Nup157, and Nup170, as
well as the extreme C termini of Ndc1 and Pom34 (Fig. 1).
little regular secondary structure.
The 44 nup domains were assigned to only eight fold types (Fig.
1 and Table 1): the ?-solenoid fold covers the most residues (38%).
The next most prevalent are the FG repeats (29%) and the
?-propeller (16%) fold. Each of the other five fold types [the TMH
fold, the cadherin fold, the coiled-coil fold, the autoproteolytic
Nup98 domain, and the RNA recognition motif (RRM)] individ-
ually covers ?5% of the total nup residues (Table 1).
In an effort to substantiate the yeast nup fold assignments and
provide a complete structural coverage of the nups from yeast to
vertebrate, we also assigned folds to the Rattus norvegicus nups (4)
as well as their predicted Homo sapiens homologs. Although these
orthologs generally share ?40% sequence identity, their sequence
similarity is statistically significant (i.e., the BUILD?PROFILE E value
is ?0.01). Fold assignments for nups from vertebrates matched
those for their yeast homologs (Table 2, which is published as
supporting information on the PNAS web site).
Assessment of Fold Assignments. Our fold assignments are sup-
ported by seven considerations. First, for 16 of the 19 domains
assigned by threading (Table 1), the assignment was statistically
significant as defined by the authors of the threading methods
(18–21) for at least three of the four methods. In addition, no other
fold type occurred as frequently in the list of significant hits for any
of the assigned domains. Moreover, although there are numerous
sequence and structure variations among proteins of the same fold,
the different servers often selected the same template structures.
The folds for the remaining three domains are supported by other
considerations (see below). Second, despite their low sequence
similarity, the human orthologs of the yeast nups independently
share their fold assignments without exception (Table 2). Third,
of the nups. The names of the nups are boxed accord-
ing to the group they define: transmembrane (pink),
scaffold (orange), and FG repeat (green). The black
?-strands (cyan) are indicated by bars above each line.
The height of the bars is proportional to the confi-
dence of the prediction (39). Predicted transmem-
brane helices are shown in green, coiled coils are
shown in red, FG repeats are shown in black, and
unstructured regions are shown by an empty box. An
orange block underlines regions of ?50 residues to
which a fold type could not be assigned. Representa-
tive models of the nup domains are colored according
to the fold type and are shown on the left. Models are
not to scale for visualization reasons. There are eight
bic 15- to 30-residue helical segment that spans the
membrane. Second, cadherin domains (dark blue)
have ?110 residues that fold into a seven-stranded
?-sandwich structure. Third, ?-propellers (cyan) con-
tain several blades arranged radially around a central
axis, each blade consisting of a four-stranded antipa-
rallel ?-sheet. Fourth, ?-solenoid domains are com-
posed of numerous pairs of antiparallel ?-helices
stacked to form a solenoid. Fifth, coiled coils (red)
and fourth residues of an ?-helix are often hydropho-
bic. The coiled-coil structure is formed by helices (gen-
erally two) twisting together to bury their hydropho-
bic seams. Sixth, disordered FG-repeat segments are
indicated schematically by a black curve. Seventh, the
autoproteolytic domain of Nup98 (yellow) adopts a
half-open, ?-sandwich-like fold dominated by a large
?-sheet with helices capping two of its ends. Finally,
found in proteins involved in RNA binding.
Predicted secondary structure and fold types
Devos et al.PNAS ?
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assessment scores for comparative models based on each fold
assignment were statistically significant when compared against the
best models generated for random sequences of identical amino
acid residue composition and length; all of the nup models were at
least four standard deviations away from the mean score of the
random models (Table 1). Fourth, secondary structure predictions
from sequences largely matched the secondary structures in the
corresponding comparative models. The agreement is as high as
87% of the residues for some of the models, when a three-state
assignment (helix, strand, and other) is used. This agreement is the
the secondary structure prediction methods (22). Fifth, protease
also Table 3, which is published as supporting information on the
PNAS web site). Limited proteolysis is expected to occur in regions
that are exposed to the solvent and outside secondary structure
segments, corresponding to domain boundaries and loops. There-
fore, protease accessibility laddering can be used to test the
predicted boundaries between domains and perhaps even regular
secondary structure segments. For the multidomain nups, the
strongest cleavage indeed occurs between the predicted domains
within a residue of an exposed boundary of a regular secondary
boundaries in models of the same sequences based on randomly
selected folds. This result is statistically significant with a P value of
and Nup133 (16) as well as Nup157 and Nup170 (Fig. 2), which are
all predicted to contain the same fold arrangement (Fig. 1 and
Table 1). Sixth, circular dichroism and Fourier-transform infrared
spectra of Nup85 are in agreement with our predictions, indicating
a composition characteristic of ?-solenoid domains (?50% ?-helix
and 10% ?-sheet) (23). Similarly, the circular dichroism spectrum
Finally, recent atomic structure determinations of the N-terminal
domains of Nup133 (14) and Nup159 (15) as well as the NPC-
for these proteins.
We assigned folds to most domains in nups from S. cerevisiae, R.
in the composition and modularity in the architecture of the NPC.
eight predicted fold types, most composed of multiple, simple
repetitive units (Fig. 1 and Table 1). The modularity of the
their predicted folds into only three groups (Fig. 3). Such simplicity
and modularity suggests that the evolution of the NPC occurred by
many intragene and full-gene duplications followed by divergence.
Simplicity of the NPC Composition.Thecompositionofthepredicted
domain folds in the yeast NPC is exceedingly simple, given that the
NPC is an ?50-MDa complex consisting of ?480 proteins of ?30
different types. The three most frequent domains (i.e., the ?-sole-
noids, FG repeats, and ?-propellers) account for 83% of the
residues, whereas only five further fold types (i.e., the TMH,
cadherin, coiled-coil, Nup98, and RRM folds) account for most of
the remainder. The increased proteolytic susceptibility of the
unassigned fragments suggests that they are enriched in unstruc-
tured and?or flexible regions. Therefore, with a possible exception
and Pom34, it is likely that folds for all domains with defined
structure have been detected. Thus, the conclusion that a small
number of repeat elements is sufficient to build the NPC is unlikely
of the NPC must include membrane proteins for the anchoring of
the NPC into the NE, the scaffold proteins that provide the
framework for assembling the NPC, and the selective filter that
presumably lines the central passage through which the transport
occurs (1, 25). The fold assignments can be easily interpreted in
terms of such a simple architecture as follows (Fig. 3).
The first group consists of the three membrane-spanning nup
(Fig. 3b) (3). The transmembrane ?-helices of these pore mem-
they may help in stabilizing the curvature of the NE through the
Table 1. Nup domains are predicted to be clustered into eight
Shown are the number of residues in the corresponding nup sequence
(size), an estimate of the domain range, the fold type of the corresponding
model (where CC is coiled coil, FG is FG repeat, and Nup98 is the autocatalytic
domain of Nup98), and Z scores of the comparative models (46) for the
residues indicated in the parentheses. The predicted TMH, FG repeat, and
coiled coil folds were not modeled. n.a., not applicable.
*The value in parentheses is the count of TMHs.
†Indicates that the nup is characterized in ref. 16.
www.pnas.org?cgi?doi?10.1073?pnas.0506345103Devos et al.
cadherin domains of Pom152. The cadherin domain has been
observed to provide a bridge between two membranes (e.g., in
desmosomes) (26). It is conceivable that it plays an equivalent role
in stabilizing the interaction between the outer and inner nuclear
membranes of the NE.
The second group is formed by the nups predicted to contain
primarily the FG repeat and coiled-coil domains, in addition to the
infrequent Nup98 and RRM folds (Fig. 1). The FG repeats were
shown to provide low-affinity, high-specificity interactions with
transport factors involved in active transport through the NPC (27)
coats the central pore surface (Fig. 3b), providing an interaction
region for transport factors around the central channel of the NPC.
Moreover, the frequent mediation of protein interactions through
coiled coils (28) suggests that the coiled-coil domains anchor the
FG nups into the bulk of the NPC.
The third group consists of the nups predicted to contain only
?-solenoid and ?-propeller folds (Fig. 1). Every nup without a
transmembrane helix, FG repeat, or coiled-coil domain contains
the ?-solenoid or ?-propeller or both folds. Given the proposed
anchoring the two groups to form a complete NPC (Fig. 3b). This
proposal is further supported by our previous observation relating
the seven nups in the Nup84 subcomplex to the proteins covering
the clathrin, COPI, and COPII coated vesicles (16). In coated
vesicles, the clathrin-like and adaptor proteins constitute the struc-
tural scaffold of the protein coat surrounding the membrane of the
vesicle in a lattice fashion (29), perhaps similarly to the role of
the scaffold nups in the NPC. We extend the original proposal [the
the scaffold nups and the coated vesicle proteins originated from a
common precursor complex (i.e., the protocoatomer). Both ?-
solenoid and ?-propeller folds provide extensive solvent-accessible
surfaces that appear well suited for binding other proteins. More-
over, the ?-solenoid and ?-propeller folds seem to be sufficiently
robust for significant variation in sequence while retaining the core
structure, thus allowing optimization of interactions with a multi-
tude of other partners.
The fold assignments for the nups, therefore, (i) reinforce the
protocoatomer hypothesis (16), (ii) extend the hypothesis to all of
the structural core of the NPC, and (iv) further indicate that same
Evolution of the NPC. The simplicity of the putative NPC compo-
sition and architecture also suggests how the NPC evolved. The
and ?-propeller) are composed of repeat motifs. This observation
accessibility laddering. The gels show immunoblots of limited
proteolysis digests for Protein A-tagged versions of the nups.
Each full-length nup is indicated by an arrow on the left side
of the gel, as are the sizes of marker proteins (expressed in
kDa). Secondary structure predictions are shown by using the
conventions in Fig. 1. Proteolytic cleavage sites are identified
by small, medium, and large arrows for weak, medium, and
strong susceptibility sites, respectively. Where necessary, un-
certainties in the precise cleavage positions are indicated by
lines to the left of the sequence.
Mapping of domains of the yeast nups by protease
Devos et al. PNAS ?
February 14, 2006 ?
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no. 7 ?
indicates extensive intragenic duplication in the evolution of the
NPC and is consistent with a prior observation that repeat proteins
play an important role in eukaryotic evolution in general (30). In
folds with repeats, the number of repeats can vary even between
orthologs, indicating that rapid loss and gain of repeats occur
frequently. Indeed, such variation in repeat number is seen for the
FG repeats of Nsp1, even in different strains of the same species
(31). Sequence similarity among repeats may erode quickly, which
makes the reconstruction of the evolutionary trajectory difficult
despite common features in sequence and structure.
The eight predicted fold types of the 44 assigned domains
comprising the 28 yeast nups indicate extensive gene duplication
and structural redundancy in the building blocks of the NPC; all of
the nups presumably originated from a minimal set of precursor
proteins by extensive intragenic and intergenic duplication events.
In fact, the same eight fold types are also found in the nups from
H. sapiens. Interestingly, additional domains are found in higher
eukaryotic nups. These predicted domains include zinc finger, Ran
binding, Ran GAP (a GTPase activating domain for Ran), and
cyclophilin domains. Many of these domains occur within a single
protein, Nup358, a 3,224-residue vertebrate protein (32, 33). These
domains constitute additional elaborations to the core NPC that
occurred during the diversification of eukaryotes.
Karyopherins. Karyopherins mediate the specific transfer of cargo
nups (27). The crystallographic structures of karyopherins show
that they assume an ?-solenoid fold (34). We have proposed here
that approximately one-third of the nups also contain ?-solenoid
domains. Moreover, these ?-solenoid-containing nups, like other
mobility is linked with the targeting of the ?-solenoid-containing
nups to more than one kind of structure in the cell; for example, a
complex of several ?-solenoid-containing nups localizes to kinet-
ochores during mitosis (36), perhaps reflecting an ancient associ-
ation between kinetochores and the NE that is still seen in
dinoflagellates (37). Given these considerations, it is tempting to
suggest that the karyopherins and the ?-solenoid nups may share
the same ancient precursor. According to this proposal, karyo-
pherins evolved from the nups that were an integral structural part
of an early NPC but diverged so that the interaction became
transient as required by a developing cargo transport function.
Evolution and Emergence. The concept of emergence suggests that,
in a structured system, at each level, units associate to form a unit
of the level above (38). Each new unit formed by the integration of
subunits from the level below has ‘‘emerged’’ characteristics and
capacities not present at any lower level of integration. This
emergence can be observed in the NPC model, in which individual
sequence motifs multimerize to form much larger proteins (Fig. 3a,
step I) that assemble into multiple copies to form each half-spoke
(Fig. 3a, step II) that eventually dimerize to form the spokes (Fig.
3a, step III), which are themselves repeated eight times to form the
complete NPC (Fig. 3a, step IV). Thus, the entire NPC appears to
be based on the hierarchical repetition of simple structural motifs
to form an elaborate structure. In addition, it is reasonable to
of sequence repeats (step I). The nups assemble into multiple copies to form each half-spoke (step II) that dimerize to form the spokes (step III), which are
themselves repeated eight times to form the complete NPC (step IV). (b) The architecture of the NPC ring, viewed in the plane of the NE, is segregated into the
membrane (pink), scaffold (orange), and FG (green) groups. The domain fold types assigned to each group are indicated on the left side of the schema. The
schema illustrates the coarse organization of the NPC and is not a precise map of the three-dimensional nup locations.
Simplicity of the fold composition and modular architecture of the NPC. (a) The schematic structure and hierarchy of the NPC. Most of the nups consist
www.pnas.org?cgi?doi?10.1073?pnas.0506345103Devos et al.
suggest that this hierarchical structure evolved through a series of Download full-text
gene duplications and divergencies.
We find simplicity at several levels in the NPC architecture. There
are only eight predicted fold types assumed by the domains of 28
of repeats of regular secondary structure segments. This compo-
the evolution of the NPC. Thus, the entire NPC appears to be
3b). It is conceivable that even karyopherins, which transiently
interact with the NPC during their mediation of the transport
through the pore, evolved via duplication and divergence from the
ancestors common to both karyopherins and scaffold nups. It
remains to be seen whether the three-dimensional structure of the
NPC, once solved, reflects the simplicity of the architecture sug-
gested by the proposed fold assignments.
Materials and Methods
Predicting Sequence Features of Nups. The secondary structure,
TMH, disordered, and coiled-coil regions were predicted from
sequence by the PSI-PRED (39), PHOBIUS V2.0 (40), and DISOPRED
servers (41) and the COILS program (28), respectively. FG repeat
domains were identified as the disordered regions containing
repeats of any of the following sequence motifs: FG, FxF, FxFG,
GLFG, SAFG, PSFG, and SAFGxPSFG, where we allowed x to be
any residue type.
Sequence-Based Searching for Nup Homologs. Disordered segments
were excluded from sequence comparisons, because they tend to
fail the assessment of statistical significance of a sequence match.
The nups were divided into domains by an iterative manual process
alignments, and sequence–structure alignments from threading.
For each nup domain, homologous sequences in the nonredundant
UNIPROT90 database (42) were detected by the BUILD?PROFILE
command of MODELLER-8 (43). BUILD?PROFILE is an iterative
database-searching tool that relies on local dynamic programming
to generate alignments and a robust estimate of their statistical
significance (44); as a result, BUILD?PROFILE detects ?50% more
scanned against our database of profiles for the domains of known
structure, using the PP?SCAN command of MODELLER-8. PP?SCAN
compares pairs of sequence profiles by a local dynamic program-
ming algorithm using the correlation coefficient between profile
columns as the scoring function.
Fold Assignment by Threading and Model Assessment. Nupsegments
unmatched to a known structure by the sequence-based searches
were submitted to the MGENTHREADER (18), FUGUE (20), AGAPE
(19), and HHSEARCH (21) fold assignment web servers. We com-
bined these results into consensus fold assignments and evaluated
the fold assignments by an iterative process of alignment, compar-
ative model building, and model assessment (16).
Protease Accessibility Laddering. We performed proteolytic domain
mapping (16, 47) for nine yeast nups. Together with the previous
mapping of the seven nups in the Nup84 subcomplex (16), we thus
cover all nups without FG repeats. The protease accessibility
laddering data were used to assess the fold assignments. Quantifi-
cation of the agreement between the observed proteolytic cuts and
the model is based on the assumption that the proteases cut only
next to specific residues (i.e., Lys, Arg, and Asp) that are exposed
to the solvent and located between secondary structure elements
(i.e., exposed loops). Each model was assessed as follows. We
compared the number of experimentally observed cleavage loca-
tions occurring in exposed regions between secondary structure
segments of the model with the corresponding number expected by
chance. This latter number was obtained by modeling the sequence
using randomly selected folds as templates.
We thank the members of the A.S. and M.P.R. laboratories for discus-
sions about the NPC, especially Maya Topf and Fred Davis. We also
thank Joe Fernandez and the Proteomic Resource Center of The
Rockefeller University for protein sequence analysis. This work was
supported by The Sandler Family Supporting Foundation, Sun Micro-
systems, IBM, Intel, and National Institutes of Health Grants GM62529
and GM54762 (to A.S.); by an Irma T. Hirschl Career Scientist Award,
a Sinsheimer Scholar Award, a grant from the Rita Allen Foundation,
and National Institutes of Health Grants GM062427 and RR022220
(to M.P.R.); and by National Institutes of Health Grants RR00862
(to B.T.C.) and CA89810 (to B.T.C. and M.P.R.).
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