Title:
Architecture of the cytoplasmic face of the nuclear pore
Authors:
Christopher J. Bley,
1#
Si Nie,
1#
George W. Mobbs,
1#
Stefan Petrovic,
1#
Anna T. Gres,
1#
Xiaoyu Liu,
1#
Somnath Mukherjee,
2
Sho Harvey,
1
Ferdinand M. Huber,
1
Daniel H. Lin,
1
Bonnie Brown,
1
Aaron W. Tang,
1
Emily J. Rundlet,
1
Ana R.
Correia,
1
Shane Chen,
3
Saroj G. Regmi,
3
Mary Dasso,
3
Alina Patke,
4
Alexander F. Palazzo,
5
Anthony A. Kossiakoff,
2
André Hoelz
1
*
Affiliations:
1
California Institute of Technology, Division of Chemistry and Chemical Engineering, 1200 East California
Boulevard, Pasadena, CA, 91125, USA
2
Department of Biochemistry and Molecular Biology, The University of Chicago, Chicago, IL 60637, USA
3
Division
of Molecular
and Cellular Biology
, National Institute of Child Health and Human Development,
National Institutes of Health, Bethesda, USA
4
California Institute of Technology, Division of Biology and Biological Engineering, 1200 East California
Boulevard, Pasadena, CA, 91125, USA
5
Department of Biochemistry, University of Toronto, Toronto, Ontario, Canada
#
these authors contributed equally to this work
*Correspondence: hoelz@caltech.edu (A.H.)
1
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint
this version posted October 26, 2021.
;
https://doi.org/10.1101/2021.10.26.465790
doi:
bioRxiv preprint
Abstract:
The nuclear pore complex (NPC) is
the sole
bidirectional
gateway for
nucleocytoplasmic transport
.
Despite recent progress in
elucidating
the NPC symmetric core
architecture, the
asymmetrically
decorated cytoplasmic
face,
essential
for mRNA export
and a hotspot for nucleoporin-
associated
diseases, has remained elusive.
Here, we
report
a composite structure of the entire human cytoplasmic
face obtained
by combining biochemical
reconstitution,
crystal structure determination
, docking into
cryo -electron tomographic reconstructions
, and
physiological
validation, accounting for
a third of the
NPC’s mass.
Whereas an
evolutionarily conserved ~540
kDa hetero
-hexameric cytoplasmic filament
nucleoporin complex is anchored by species
-specific
motifs
above
the central transport channel,
attachment of the pentameric NUP358 bundles depends on the double
-ring arrangement of the coat
nucleoporin complex.
Our results
and the predictive power of our composite structure provide a rich
foundation for
elucidating
the molecular
basis
of mRNA export and nucleoporin diseas
es.
One sentence summary:
An interdisciplinary
analysis established the near
-atomic
molecular architecture
of the
cytoplasmic face
of the
human
nuclear pore complex.
2
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint
this version posted October 26, 2021.
;
https://doi.org/10.1101/2021.10.26.465790
doi:
bioRxiv preprint
Introduction
The sequestration of genetic material in the nucleus represents one of the great hallmarks of evolution
but
creates the necessity for selective bidirectional transport across the nuclear envelope
(
1-4
). The nuclear
pore complex (NPC) is the sole gateway through which folded proteins and protein/nucleic acid complexes
cross the nuclear envelope, making this transport organelle an essential machine for all eukaryotic life.
Besides its direct role as a trans
port channel, the NPC serves as an organizer for nuclear and cytoplasmic
processes that are essential for
the flow of genetic information from DNA to RNA to protein, including
transcription,
spliceosome assembly,
mRNA export
, and ribosome assembly
(
1-4
). D ysfunction of the NPC
or its components represents a major cause of human disease
(
2, 5 , 6
).
Architecturally, the NPC consists of a central core with an 8
-fold rotational symmetry across a
nucleocytoplasmic axis and a two-
fold rotational symmetry across the plane of the
nuclear envelope, which
links to compartment
-specific asymmetric
“cytoplasmic filaments” (CF)
and a “nuclear basket” structure
(Fig. 1A) (
1, 2
). The NPC is built from ~34 different protei
ns, termed nucleoporins (nups) that are organized
into distinct subcomplexes.
Multiple copies of each
nup in the NPC
add up to an
assembly
that reaches
an
extraordinary molecular mass of ~1
10 MDa in
vertebrates.
The symmetric core
of the NPC
is composed
of an inner ring and two spatially segregated outer rings
. The inner ring is embedded in nuclear envelope
pores generated by the circumscribed fusion of the double membrane of the nuclear envelope.
The
diffusion barrier is formed by unstructured phenylalanin
e- glycine
(FG) repeats
that fill the central transport
channel
, imposing
an ~40
kDa limit on passive diffusion
(
1-4
). Transport factors collectively termed
karyopherins overcome the diffusion barrier by binding to FG repeats, thereby transporting cargo across
the nuclear envelope
(
7-9
). A significant fraction of the FG
repeat
s in the in
ner ring
is contributed by
a
hetero-
trimeric
channel
nup complex
(CNT) which is anchored by a single assembly sen
sor motif (
10-
12
). The outer rings sit atop the nuclear envelope
, sandwiching the inner ring from both sides. They
are
primarily formed by the Y-
shaped coat nup complex (CNC)
and serve as a platform for the asymmetric
incorporation of the
CF and nuclear basket nups.
Two decades
ago, the atomic level characterization of the NPC began with individual nup domains
and progressed to nup complexes
of increasing
size and complexity,
culminating in
the ~400 kDa
3
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint
this version posted October 26, 2021.
;
https://doi.org/10.1101/2021.10.26.465790
doi:
bioRxiv preprint
hetero-
heptameric Y
-shaped
CNC
(
11, 13- 28
) . Simultaneously, advances
in the acquisition
and
processing of
cryo -electron
tomographic (cryo
-ET) data gradually increased the resolution of intact NPC
3D reconstructions
(
29
). Docking
of the CNC into a ~32
Å cryo
-ET map of the intact human NPC
demonstrated that two reticulated eight
-membered CNC rings, linked by head
-to-tail interactions, are
present on each side of the nuclear envelope
(
27, 30
). Moreover, this advance established that the
resolution gap between high
- and low
-resolution structural methods can be overcome by combining
biochemical reconstitution
and X-
ray crystallographic charact
erization
of nups
with
cryo -ET
reconstruction of the intact NPC
. Expansion
of this approach to the nine
nups constituting the inner ring
rapidly led to the reconstitution of two distinct ~425 kDa inner ring complexes and the elucidation of their
components’ structures
(
10- 12, 20, 31- 38
).
In turn, this advance enabled the determination of the
near-atomic composite structure of the entire
~56 MDa symmetric core of the human NPC, establishing
the stoichiometry and placement of all 17 symmetric nups within a ~23
Å cryo-
ET reconstruction
(
38, 39
).
Subsequently, the architecture of the
Saccharomyces
cerevisiae
NPC was determined with
a similar
approach, utilizing high
-resolution nup crystal structures and ~25
Å cryo
-ET maps of either detergent
purified or
in-situ
NPCs
(
40, 41
). Compared to the human NPC,
the
S. cerevisiae
NPC l
acks
the distal
CNC ring
and associated
nups
on both sides of the
nuclear envelope
, but the relative nup arrangement
within the rest of the symmetric core remains
essentially identical
(
38, 39, 42
).
Projecting into
the cytoplasm from the cytoplasmic face of the NPC, the
CF nups
recruit
cargo
•transport factor complexes
for nucleocytoplasmic transport
and orchestrate the export and
remodeling
of messenger ribonucleoprotein particles (mRNPs)
in preparation for translation (
2, 43
). The
nine-component
CF nup
machinery represents a major hotspot for
human
diseases
ranging from
degenerative brain disorders and
cardiac diseases to
cancer
(
2, 5, 6
). Although linked to the human CF
nups
NUP358, NUP214, NUP62, NUP88, NUP98, GLE1, NUP42, RAE1, and DDX19
, the pathophysiology
and optimal therapeutic strategies
for these conditions
remain ill
-defined.
Here,
we present
insight into the
atomic and higher order architecture, function,
and mechanism
of action of the CF nups
in the
human NPC and the NPC of the thermophilic fungus
Chaetomium
thermophilum
.
First, we uncover a
conserved
modular architecture within
the hetero-
hexameric
CF nup
4
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint
this version posted October 26, 2021.
;
https://doi.org/10.1101/2021.10.26.465790
doi:
bioRxiv preprint
complex (CFNC)
of both species: Holding the CFNC together is a coiled
-coil hub built like the
CNT, but
formed by NUP62 with the C
-terminal regions
of NUP88
and NUP159,
while their
N- terminal
b
-propell
er
domains
link to
the mRNA export factor
s NUP98
•
RAE1
and the DEAD-
box RNA helicase DDX1
9,
respectively, which in turn recruit the remaining
complex components
. We further uncover evolutionary
divergent mechanisms for the attachment of the intact CFNC at the cytoplasmic face of the NPC, which
in
C. thermophilum
involve
s two distinct assembly sensors in the CNC
that do not
exist in humans.
We
are able to assemble the
C. thermophilum
CNC and CF nups into a
~1.1
MDa 16-
protein complex and
find that it can be remodeled by
inositol hexaphosphate
(IP
6
). Towards dissecting the molecular
mechanism of mRNA export, we systematically characterize
the propensity of CF nups for
RNA binding
and find novel capabilities in two CFNC subcomplexes
GLE1
•NUP42 and NUP88
•NUP214
•NUP98 as
well as different parts of the metazoan
specific NUP358.
To build a composite structure of the human
NPC
cytoplasmic face, we
determine crystal structures of the NUP88
NTD
•
NUP98
APD
complex and all
remaining structurally uncharacterized regions
of NUP358, uncovering a hereto unobserved, S
-shaped
fold of three
a
-helical solenoids for the NUP358 N
-terminal domain as well as a complex
mechanism for
NUP358 oligomerization.
D ocking of
the novel structures along with previously
characterized
CF nup
s
into a previously
reported ~2
3 Å and an unpublished ~12 Å cryo-
ET map
of the intact human NPC
(provided by the Beck group),
as well as
an ~8 Å region
of an anisotropic
single
particle cryo-
EM
composite map
of the
Xenopus
laevis
cytoplasmic
NPC
face account
s for all
of the asymmetric density
on the cytoplasmic
NPC-
side resolved in the maps
(
44- 46
). V
alidat
ing our quantitative docking analysis
in human cells engineered to enable rapid, inducible NUP358 depletion, we surprisingly find NUP358 to
be dispensable for
the architectural integrity of the
assembled
interphase NPC
and mRNA export but
having a general role in translation
. The docking of the CFNC
-hub in close proximity
to a NUP93
fragment
that, in the inner ring, acts as the assembly sensor for the CNT
, allows us to predict and experimentally
confirm
that NUP93 also recruits
the structurally related CFNC on the cytoplasmic face, thereby enabling
identification of the elusive human CFNC NPC anchor.
Thus, our near
-atomic composite structure
possesses predictive power, demonstrating
its general utility for the mechanistic dissection of essential
cell ular events occurring on the cytoplasmic face of the human NPC.
5
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint
this version posted October 26, 2021.
;
https://doi.org/10.1101/2021.10.26.465790
doi:
bioRxiv preprint
RESULTS
Modular
architecture of the evolutionar
ily conserved
6- protein
cytoplasmic filament nup complex
Although pair
-wise
interactions
between selected CF nups had
previously been reported, comprehensive
knowledge on the entire CF nup interaction network has remained unavailable to date
(
11, 47- 64
). We
had previously
found that nups from
C. thermophilum
exhibit
superior biochemical stability
, allowing us
to overcome long
-standing technical challenges and
elucidate the interaction network of the
seventeen
symmetric
core nups (
38
). We therefore
first sought to establish the protein
-protein interaction network
and complex stoichiometry
of the eight evolutionarily conserved
C. thermophilum
CF nups Nup82,
Nup159, Nsp1, Nup145N, Gle2, Dbp5, Gle1, and Nup42
(
2
) ( Fig. 1B and fig.
S1). Most
CF nups
contain
both
structured
and unstructured
regions
that can
harbor
multiple
distinct binding sites
and FG
repeats
(Fig. 1B).
We established expression and purification protocols for
the
C. thermophilum
CF nups, omitting
FG- repeat regions as well as an unstructured linker region in Nup145N t
o improve
solubility, and analyzed
their binding
by size- exclusion
chromatography coupled with multiangle light scattering (SEC
-MALS)
(fig s. S1 to
S 3 and tables S1 to S5).
Nup82
and
Nup159 both contain N
-termin
al
b
-propeller domains and C
-terminal tripartite
coiled-
coil segments (CCS1
-3), which have previously been
shown to complex with the similarly built
C- terminal coiled-
coil region of Nsp1
(
49, 65
). Another complex has previously been shown to be formed
between the
Gle2
b
-propeller and the
Gle2 -binding sequence (GLEBS)
motif
of Nup145N
(
48, 57
). Mixing
of the Nup82•
Nup159
•Nsp1 and
Gle2 •Nup145N complexes with
the DEAD-
box RNA helicase Dbp5
resulted in a
stoichiometric hetero
-hexameric
complex that we will refer to as
the cytoplasmic filament
nup complex (CFNC)
( Fig. 1
, C and D
and fig. S4
A and table S6
). To further map CF nup interactions
within this complex, we systematic
ally removed
individual fragments and d
omains from the CFNC and
probed
for remaining interactions
( fig. S
4). Consistent with previous
characterizations
of isolated CF nup
pairs and trimers (
11, 47- 64
) , this analysis established that the
N- terminal
b
-propeller domain of Nup82
binds an
a
-helical TAIL segment in Nup159 and
the autoproteolytic domain (ADP) of Nup145N, which in
turn recruits Gle2
( Fig. 1C
and fig.
S4) . Analogously, the Nup159 N
-terminal
b
-propeller domain provides
the binding site for the Dbp5 (
Fig. 1C
and fig.
S4 ). These results
suggested that
the CFNC
must be
held
6
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint
this version posted October 26, 2021.
;
https://doi.org/10.1101/2021.10.26.465790
doi:
bioRxiv preprint
together
by the C-
terminal coiled-
coil regions of Nup82, Nup159, and Nsp1. Indeed, when the CFNC is
divided into
f our distinct
stable subcomplexes,
the Nup82•Nup159•Nsp1
C- terminal
coiled-
coil hetero-
trimer, Nup82
NTD
•Nup159
TAIL
•Nup145N
APD
, Nup159
NTD
•Dbp5, and Gle2•Nup145N
GLEBS
, their mixing did
not result in any additional interactions and
the four subcomplexes
did not bind
each other
, demonstrating
that the
entire
CFNC is held together
by interactions in the
Nup82•Nup159•Nsp1
C- terminal
coiled-
coil
heterotrimer (fig. S
5). In addition to the CFNC, Nsp1 is also an essential part of the
hetero-
trimeric
CNT,
where it
form s a parallel array of three coiled-
coil segments
together
with Nup49 and Nup57
(
10, 11
).
Given
these similarities,
we wondered whether the
coiled-
coil segments of
Nup82•Nup159•Nsp1 also
formed
a parallel coiled-
coil domain.
Indeed, we
were able to
reconstitute
separate stochiometric
complexes of the CCS1 and the CCS2-3
coiled-
coil regions of
Nup82•Nup159•Nsp1
, consistent with the
CCS1-3 coiled-
coil regions of Nup82, Nup159, and Nsp1 forming a CFNC
-hub similar to the CNT
(fig. S
6).
Having
characterized the protein
-protein interactions within the CFNC, we
next tested whether
the reconstituted complex was capable of binding to the
hetero-
octameric
CNC
(Nup120•Nup37•Elys•Nup85•Sec13
-Nup145C•Nup84•Nup133
D
NTE
), its neighbor in the intact NPC
(fig. S7
). Indeed, the reconstituted
CFNC and CNC
form ed a stoichiometric
1:1 complex
that was
dependent on the
CFNC-
hub (
fig. S8
). Because
the structurally
related
CNT
is recruited to
NPCs
through
a 39- residue
a
-helical
assembly sensor
supplied by
Nic96
(11
) , we next asked whether the CNC harbors
equivalent assembly sensors for the CFNC-
hub.
Among
the CNC
subunits
, Nup133, Nup145C, Nup85,
and Nup37
each have N-
or C- terminal
primarily unstructured extensions
(NTEs, CTEs)
that could
potentially
contain
assembly sensors
and were tested for binding
to the
CFNC-
hub. This systematic
analysis
identified
Nup37
CTE
and Nup145C
NTE
as binding partner
s ( fig. S9
). Deletion of the CTE
from
Nup37 abolished its binding to the CFNC
-hub showing that Nup37
CTE
is both necessary and sufficient for
binding (
fig. S10 ). Moreover,
removal of Nup37
CTE
from the intact CNC
substantially
reduce
d its
interaction
s with the CFNC
( fig. S11A
). While the removal of Nup145C
NTE
alone
from the CNC
had no
detectable effect,
simultaneous
elimination
of both Nup37
CTE
and Nup145C
NTE
almost
completely
abolishe
d the interaction with CFNC
(fig. S11 , B and C
). Preferential CFNC-
hub recruitment by Nup37
CTE
7
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint
this version posted October 26, 2021.
;
https://doi.org/10.1101/2021.10.26.465790
doi:
bioRxiv preprint
persisted
when
the CNC
was dissected into
its three subcomplexes, with residual and stoichiometric
interactions formed
with
Sec13
•Nup145C
and
Nup120•Nup37•Elys•Nup85
, respectively
( fig. S12 ).
Vestigial CFNC binding
to a CNC lacking both Nup37
CTE
and Nup145C
NTE
was mapped to a weak CNC
-
interaction with Gle2
-Nup145N
GLEBS
( fig. S13 ).
In the
CNT•
Nic96 crystal structure
, the 39- residue
Nic96
assembly
sensor
forms
two ~
10- residue
a
-helical segments
joined
by an ~
15- residue
linker
, with the C
-terminal
helix being
necessary and
sufficient for CNT binding
(11
). S
econdary structure predictions
for both Nup37
CTE
and Nup145C
NTE
suggest
comparable arrangements
of two shorter
a
-helices
separated by a linker
from
a longer
a
-helix.
Moreover,
Nup145C
NTE
interactions have previously been mapped to an
equivalent region
,
encompassing the C
-terminal helix followed by a short unstructured segment
(
62
). Through
systematic
truncation of
Nup37
CTE
, we mapped its
minimal
CFNC-
hub binding
site to the predicted C-
terminal helix
and a short unstructured
region (
fig S14 ). This demonstrates
that the
predicted
C- terminal
a
-heli x in both
Nup37
CTE
and Nup145C
NTE
assembly sensors
is necessary and
sufficient
for CFNC
-hub binding
, whereas
the N-
terminal helices likely
adopt an organizational role
in stabilizing a
specific
tertiary structure of
the
three coiled-
coil segments
, as previously observed for the CNT
-Nic96 interaction
(11
).
Next, we asked whether Nup145C
NTE
and Nup37
CTE
function
as
bona
fide
assembly sensors for
the CFNC
-hub by recognizing
intact
hetero-
trimeric coiled
-coils but not
isolated CFNC-
hub constituents
.
Pulldown
experiments
probing Nup37
CTE
or Nup145C
NTE
with
bacterial lysates
containing
individual
CFNC-
hub component
s or a mixture of all three
, revealed that
both fragments bound exclusively
to the
assembled
hetero-
trimeric
CFNC-
hub, but failed to recognize individual component nups (
fig. S15,
A and B
). Moreover,
splitting
the CFNC
-hub into CCS1 and CCS2
-3 portions
reveal
ed that both Nup37
CTE
and Nup145C
NTE
bind preferentially to
CCS2-3,
albeit weaker than to the
intact
CFNC-
hub CCS1-3
(fig s. S6
and S15C
).
Bec
a
use our SEC-
MALS analysis
indicate
d that the CNC
only binds
to a single
CFNC
copy, we
investigated
whether Nup37
CTE
and Nup145C
NTE
can simultaneously bind to
the CFNC
-hub.
We found
that Nup37
CTE
and Nup145C
NTE
bind the CFNC
-hub in a mutually exclusive manner
, via a hydrophobic
interface
stable in
a high salt (1
M NaCl ) buffer (
figs. S15D
and S16).
In fact, Nup37
CTE
outcompetes
8
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint
this version posted October 26, 2021.
;
https://doi.org/10.1101/2021.10.26.465790
doi:
bioRxiv preprint
Nup145C
NTE
for CFNC
-hub binding,
consistent with
both our
intact
CNC
and CFNC
interaction
experiments (
fig s. S12 and S17
). Although
we conducted
our SEC-
MALS analyses at the
highest
feasible
concentrations, we
acknowledge
the apparent discrepancy arising
from the detection of a single
CFNC
binding
to an intact
CNC
that harbored
two assembly sensor
binding sites
, yet at
permissible
concentrations
Nup145C
NTE
interactions are unstable
.
In addition to the hetero
-hexameric CFNC
analyzed thus far, Gle1 and Nup42 locate to the
cytoplasmic
face of the NPC, where they are involved in the export and remodeling of mRNPs
(
63
) . Gle1
can interact with Dbp5 in
an IP
6
-dependent manner and also binds Nup42 at its Gle1
-binding motif (GBM)
(
53, 58, 63, 66
). To further characterize nup complex interactions at the cytoplasmic NPC side
, we tested
for incorporation of
Gle1 •Nup42
GBM
into the CFNC or CNC.
Despite
binding only weakly to
its known
interaction partner
Dbp5 in isolation
, Gle1•
Nup42
GBM
form ed a stable,
stochiometric
, hetero-
octameric
complex with the
CFNC
that cannot be mapped to any of its sub
-complexes, indicating a
distributed
binding site (
Fig. 1E
and figs. S
18 to S21 ). Furthermore,
Gle1•
Nup42
GBM
alone does not interact
with the
CNC,
but it can be incorporated into the CNC
•CFNC c
omplex, forming a
stoichiometric
~1.2 MDa
16- protein nup assembly (
Fig. 1
F and
fig s. S22 and S23A).
Because the Gle1
-Dbp5 interaction is
IP
6
-dependent
(
53, 66
) , we carried out all SEC-
MALS interaction experiments involving Gle1•Nup42
GBM
in the presence of IP
6
. However, when
Gle1 •Nup42
GBM
, CNC, and
CFNC were
mixed in the absence of
IP
6
, the resulting
complex
displayed a molecular mass of
~2.0 MDa , consistent with the formation
of a
stochiometric dimer of the 16
-protein complex (
fig. S23B
). We systematically
mapped
this biochemical
behavior and identified a novel interaction between Gle1
CTD
•Nup42
GBM
and the CNC
, establishing that
the CNC-
CF nup interaction network can be remodeled by IP
6
( figs. S23 to S 25).
For the Y-
shaped CNC
, we had previously observed
liquid
-liquid phase separation (
LLPS)
when
subcomplexes
representing
its upper arms (
Nup120•Nup37•Elys•Nup85•Sec13•Nup145C
, CNC-
hexamer) and
base (
Nup84•Nup133
) were
mixed
(
38
). This LLPS
formation
depended
on the N- termin
al
extension (NTE)
of Nup133
, which
mediates
head-
to-tail
trans
-
interactions between CNCs
(
21
). In fact,
interaction analyses
involving CNCs
by SEC-
MAL
S
require
prevention of
LLPS
formation
by removal of
Nup133
NTE
and are also limited by an
inherent
CNC
solubility limit of
8
μ
M. To rule out
having missed
9
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint
this version posted October 26, 2021.
;
https://doi.org/10.1101/2021.10.26.465790
doi:
bioRxiv preprint
interactions
between
coat and
CF nups
in our SEC
-MALS
analyses
because of these limitations,
we
re-tested
binding behaviors by measuring the incorporation of
the respective CF nup
complexes into a
CNC LLPS condensate. Importantly, these interaction analyses performed at higher concentrations
corroborate
d all of our
SEC-
MALS
findings
(Fig. 1
, G and H,
and fig. S
26).
Given the special importance of the CF nups
for human disease, we next tested
whether
the
molecular
architecture of the CFNC
is evolutionarily
conserved
from
C. thermophilum
to humans. The
human CFNC is comprised of NUP88
(
ct
Nup82)
, NUP214 (
ct
Nup159)
, NUP62
(
ct
Nsp1), DDX19
(
ct
Dbp5), RAE1
(
ct
Gle2)
, and NUP98 (
ct
Nup145N).
Apart from a
rearrangement
of the FG
-repeat
and
coiled-
coil regions
in NUP214
, the domain organization
of the human CF
NC nups
is identical to
that of
the
C. thermophilum
orthologs (
Fig. 2
, A and B and fig.
S27).
As in
C. thermophilum
, we
developed
expression
and purification protocol
s omitting FG
-repeat and unstructured regions and analyzed
CFNC
nup interactions by SEC-
MALS. Indeed, mixing of
the NUP88•NUP214•NUP62 hetero
-trimer
with
RAE1•NUP98
and DDX19
resulted in
a monodisperse
stoichiometric ~
337 kDa
H. sapiens
CFNC
hetero-
hexamer
( Fig. 2C
and fig s. S 28 and S29). To establish
whether the
hs
CFNC
adopts
the same
modular architecture
as in
C. thermophilum
, w
e dissected
the human CF nups to
generate CFNC
sub- complexes and carried out a systematic pairwise
SEC-
MALS
interaction analysis
( Fig. 2D and
fig s. S30 to S39) . Showing complete evolutionary conservation, t
he human
CFNC could be divided into
the same four subcomplexes
we found in
our analysis of the
C. thermophilum
CF nups:
NUP88
NTD
•NUP98
APD
•NUP214
TAIL
,
NUP214
NTD
•DDX19,
RAE1•NUP98
GLEBS
,
and
the
NUP88•NUP214•NUP62 coiled
-coil complex (
hs
CFNC-
hub)
(Fig. 2D and figs.
S30 to S39). Because the
identified
Nup37 and Nup145C
coat nup
assembly sensors
for the intact
CFNC
in
C. thermophilum
each
contain
~60- residue regions with
three short
a
-helices
, we conducted
secondary structure
predictions
of
all ten human coat nups.
This
analysis confirmed the presence of extended unstructured regions in
NUP96, NUP107, and NUP133,
yet analogous assembly sensor motifs
could
not be identified.
Together, our data
establish
that the
CF nups form an evolutionarily conserved
6- protein
complex
that is
held together by an extensive
parallel
coiled-
coil hub generated by
the C-
terminal regions
of
Nu
p
82 /NUP88, Nup159/NUP214
and Nsp1
/NUP62
, which
shares
architectural similarities
with
the
10
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint
this version posted October 26, 2021.
;
https://doi.org/10.1101/2021.10.26.465790
doi:
bioRxiv preprint
heterotrimeric
Nsp1/NUP62
•Nup49
/NUP5
8• Nup57
/NUP54
CNT (
11
).
The Nup82
/NUP88
N- terminal
b
-propeller domain
is attached
by an interaction between
the C-
terminal
a
-helical TAIL fragment of
Nup159
/NUP214 and
provides a binding site for the
Nup145N/NUP98 APD, which in turn recruits
Gle2/RAE1 to the NPC
. Analogously, the Nup159/NUP214 N
-terminal
b
-propeller domain provides a
binding site for the DEAD
-box helicase Dbp5/DDX19.
In
C. thermophilum
,
the CFNC-
hub is anchored to
the CNC by two
distinct assembly sensor
s in Nup37
CTE
and Nup145C
NTE
, similar
to the
anchoring of the
CNT by the Nic96
R1
/NUP93
R1
assembly sensor in the inner ring
. In contrast,
the human CNC lacks
comparable
assembly sensor
motifs
, suggesting
alternative
mechanism
s for anchoring
CF nups at
the
cytoplasmic face
of the human NPC
.
RNA interactions
of human cytoplasmic filament nups
Given
their essential roles
in mRNA export
, w e next
sought
to identify
which of the human CF
nups
possessed
RNA-
binding
capabilities (
50, 67- 70
). Until now, the RNA binding properties of CF
nups have
been addressed by disparate
methods
, utilizing a wide but inconsistent
selection of RNA and DNA
probes, with several protein constituents
remaining
untested.
Previous
work
established
DDX19 and
RAE1•NUP98
GLEBS
binding
to U
10
single stranded ssRNA, degenerate ssRNA, poly(A), poly(C), poly(G)
RNA, as well as ssDNA and double stranded dsDNA across a variety of assays
(
50, 56, 57, 63, 71
).
Taking
advantage of our
complete set of
purified human CF nup
domains and
sub- complexes,
we carried
out a comprehensive
electrophoretic mobility shift assay (EMSA)
screen
to systematically
assess binding against a consistent set of
ss/ dsRNA probes (
Fig. 3
). We selected a 12
bp GC
-rich stem
loop capped by a UUCG tetraloop
dsRNA
and U
20
ssRNA
as generic probes.
Positive RNA interactions
where subsequently validated
by
titrating the
protein concentration
in EMSAs
to establish approximate
apparent binding constants
(
K
D
s)
(fig. S
40). Additionally, to
establish whether the observed
binding
was
RNA-
specific,
we tested
all CF nups for
binding
to ss/ dsDNA
using a
U1 3’ box ssDNA oligonucleotide in
the absence
and presence of a complimentary ssDNA oligonucleotide.
First, we assayed
the CFNC nups for
nucleic acid binding. B
oth RAE1•NUP98
GLEBS
and DDX19
bound
ss/dsRNA,
as previously shown
( fig. S40, B
and C) (
50, 57
). W
e discovered
moderate binding
of
11
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint
this version posted October 26, 2021.
;
https://doi.org/10.1101/2021.10.26.465790
doi:
bioRxiv preprint
NUP88
NTD
and
GLE1
CTD
•NUP42
GBM
to ssRNA
(Fig. 3C and fig.
S40A). I
n the context of
the
NUP88
NTD
•NUP98
APD
•NUP214
TAIL
complex,
NUP88
NTD
binding
was specifically enhanced
to ssRNA,
exhibit
ing a
K
D
≈1 50 nM compared to
K
D
s >5
μ
M for dsRNA and
ss/dsDNA probes
(fig. S40A).
In contrast,
GLE1
CTD
•NUP42
GBM
bound both
ssRNA
and ssDNA
weakly
with
K
D
s >2
μ
M. W
e found that
the
orthologous
C. thermophilum
Nup82
NTD
•Nup145C
APD
•Nup159
TAIL
, Gle1
CTD
•Nup42
GBM
, and
Gle2 •Nup145C
GLEBS
complexes
bound ssRNA,
demonstrating
that RNA binding
is an
evolutiona
rily
conserved
feature
of CFNC
nups (
Fig. 3C and
fig. S40D).
Next, we
analyzed
the RNA-
binding properties of
the metazoan
-specific
NUP358
, taking
advantage of
structurally well
-defined domain
boundaries
for the
N- terminal domain (NTD),
oligomerization element (OE)
, four
distinct
Ran- binding domains (RanBDs),
a zinc- finger
domain (ZFD)
comprising
eight C4- type
zinc- finger motifs
(ZnF)
arranged
in tandem
, and the C
-terminal prolyl
isomerase domain (CTD)
(see below)
(
72- 75
). Previously,
NUP358
ZFD
was shown to bind
to a 63 nt RNA
fragment
encoding
the signal sequence
coding region (SSCR) of insulin
, an interaction that
was
abolished in the presence of
Ran(GDP/GTP
) (
76
) . Using our four
nucleic acid
probes, we
did not detect
RNA binding for NUP358
OE
, NUP358
ZFD
, or NUP358
CTD
at the assayed concentrations
(Fig. 3A
).
However, we
detected
moderate
RNA and DNA
binding
for NUP358
NTD
( Fig. 3B and fig.
S40G
). Because
the previously identified
insulin SSCR RNA binding partner
for NUP358
was sensitive to degradation, we
systematically tested
RNAs encoding
the SSCRs
of other
secretory proteins
. This analysis
identified the
63 nt placental alkaline phosphatase (ALPP)
SSCR RNA
as most amenable to biochemical studies
(fig. S40I).
Whereas
we only observed an insubstantial shift of ALPP
SSCR
RNA with
NUP358
ZFD
,
NUP358
NTD
robustly bound to ALPP
SSCR
RNA with a
K
D
≈150 nM, ~4- fold tighter than our generic ssRNA
or dsRNA probes (
fig. S40I ). Unexpectedly, we also found that the
four NUP358
RanBD
•Ran(GMPPNP)
complexes
preferentially
bound ssRNA
, with a K
D
≈5 00 nM, over dsRNA
and ss/dsDNA
(Fig. 3B and
fig. S40E
). To test whether RNA
binding
is a general
property of
RanBD
s when bound to
Ran,
we
assayed
the related
nuclear basket NUP50
RanBD
•Ran(GMPPNP) complex,
but detected
weaker
binding
to RNA
and DNA (
fig. S40 , E and F
).
Together,
our systematic
CF nup
analysis
confirmed previously
established
RNA
binding
sites
in
12
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint
this version posted October 26, 2021.
;
https://doi.org/10.1101/2021.10.26.465790
doi:
bioRxiv preprint
DDX19, p15
•TAP, and RAE1
•NUP98
GLEBS
, whilst
uncover
ing novel
sites
in
NUP88
NTD
•NUP214
TAIL
•NUP98
APD
, GLE1
CTD
•NUP42
GBM
, NUP358
NTD
, and the
four
NUP358
RanBD
•Ran(GMPPNP)
complexes. F
uture
studies
need to delineate
whether
these RNA binding sites
present sequence specific RNA affinity
, and what the implications of such specificity would be in the
overall mRNA export pathway
.
Structural
and biochemical analyse
s of NUP358
Composed of
a 3,224-
residue polypeptide,
NUP358
is a metazoan
-specific
additional
CF component
and
the largest constituent of the NPC (
72, 75, 77, 78
). P
revious studies
established
that its N- terminal
~900-
residue
a
-helical region is necessary for
nuclear envelope
recruitment
(
79
) . Within this region
, the first
145
residues have been biochemically and structurally characterized, shown to form three
tetratricopeptide repeats (TPR)
(
80
).
Guided by secondary structure predictions
, we systematically
screened expression constructs for solubility, identifying
three
fragments: NUP358
NTD
D
TPR
(residues 145
-
752),
NUP358
NTD
(residues 1
-752), and an
extended
region
spanning residues 1
-832 (NUP358
1- 832
).
Subsequent purifications revealed that the NUP358
NTD
and NUP358
1- 832
fragments behave differently,
with the latter forming amorphous precipitates in buffer NaCl conce
ntrations
below 300
mM. Therefore,
we characterized
these
NUP358
fragments
in both high salt (350
mM NaCl) and low salt (100
mM NaCl)
buffers
, wherever possible.
NUP358
NTD
exhibited
concentration
-dependent homo-
dimerization
in low salt buffer
, with
measured molecular masses between
values corresponding to
monomeric and dimeric species
but
existed as a
monomeric species in
high salt buffer
at all
NUP358
NTD
concentrations tested
( Fig. 4
, A and
B and fig.
S41 ). Conversely, NUP358
NTD
D
TPR
was exclusively monomeric, suggesting the TPR mediates
homo-
dimerization (
Fig. 4B and fig.
S41 ). Furthermore, the extended NUP358
1- 832
fragment forms
oligomers with
measured molecular mass
es between
those of
a tetramer and a pentamer (Fig. 4C and
fig. S41 ). Subsequent C
-terminal mapping reveal
ed an oligomerization
element (OE) located between
residues 802
-832. NUP358
OE
form ed concentration
-dependent oligomers of measured molecular mass
ranging between those expected for
dimeric and tetrameric species
in both
low and high salt buffers
13
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint
this version posted October 26, 2021.
;
https://doi.org/10.1101/2021.10.26.465790
doi:
bioRxiv preprint
(Fig. 4G
and fig. S
42). Thus,
NUP358 oligomerization is mediated by
the TPR
and OE
regions,
each
located on opposite sides of the
N- terminal
a
-helical
region.
Despite extensive
efforts
, we were
initially
unable to generate
well -diffracting
NUP358
NTD
crystals
.
However, we
could obtain
crystals of
NUP358
145- 673
, allowing
for structure determination
by
single-
wavelength anomalous
dispersion
(SAD)
using
seleno-L-
methionine (
SeMet
)- labeled protein
(tables S
7 to S9)
. To aid
the crystallization
of the entire
NUP358
NTD
, we generated
high-affinity,
synthetic
antibody fra
gments
(sAB s) by phage display selection
(
81
). By systematically screening
the generated
62 sABs
as crystallization chaperones, we identified a NUP358
NTD
•sAB- 14 complex that crystallized,
enabling
de novo
structure determination
of the entire
NUP358
NTD
at a 3.95
Å resolution
( table S
10). To
unambiguously assign
the Nup358
NTD
sequence register,
we crystallized
17 additional
SeMet
-labeled
methionine mutants
( fig . S43
and t able S
11 and S12).
The asymmetric unit contained two copies of the NUP358
NTD
•sAB14 complex,
in one of which the
first three and a half
TPR
repeats
are not resolved.
The second copy forms extensive interactions with a
symmetry related molecule
( Fig. 4
, D to F, and
fig. S
44). This NUP358 NTD dimer
reveals two
alternative
TPR conformations
, in which the TPR
either
forms a continuous N
-terminal solenoid (open)
, or folds back,
separating TPR4
and forming electrostatic interactions with
HEAT repeats
5-7 of the
N- terminal solenoid
(closed)
( Fig. 4F
and fig.
S 44C).
Toggling between
these two states
provides a molecular explanation
for
the salt -sensitive, concentration
-dependent
, dimerization behavior of NUP358
NTD
( Fig. 4B
). Because
the
open confirmation is identified in the intact
NPC
(see below)
, we focus our
description on this state.
The open conf
ormation of
NUP358
NTD
can be divided in
three sections
: an N- terminal
a
-helical
solenoid composed of
four TPRs
and four H untingtin,
elongation factor 3 (EF3), protein phosphatase 2
A
(PP2A), and the yeast kinase
TOR1 (
HEAT)
repeats, a
central
a
-helical wedge domain, and a short
C- terminal
a
-helical
solenoid
formed
by three
HEAT
repeats
(fig. S
44D) . The N
- and C-
terminal
TPR
and
HEAT
repeats
ar e capped by solvating helices.
Inserted
b etween helix
a
17 of the N
-terminal solenoid
and helix
a
20 of the wedge domain
is a ~50- residue
loop that wraps
around the
convex face of the
N- terminal
solenoid.
The N-
terminal solenoid and the wedge domain form a
large
composite
concave
surface
with a striking
overall positive
charge (figs. S
45 and S46). The central wedge domain
, composed
14
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint
this version posted October 26, 2021.
;
https://doi.org/10.1101/2021.10.26.465790
doi:
bioRxiv preprint
of six
a
-helices
, makes extensive
hydrophobic
contacts with
the sides of
the N
- and C-
terminal solenoids,
generating
a non- canonical
S- shaped
overall architecture
(fig. S
44D).
Indeed, a
Dali 3D search
of the
Protein Data Bank
revealed that the NUP358
NTD
architecture is
novel
(
82
).
Our biochemical analysis revealed
that
NUP358
NTD
interacts
weakly with
NUP88
NTD
and
possesses
RNA-
binding activity,
both of which
were
susceptible
to elevated buffer
salt concentration
(figs. S
47 and S48) . By splitting NUP358
NTD
into two fragments
, NUP358
TPR
and NUP358
D
TPR
, we show
that both halves
of NUP358
NTD
are necessary,
yet insufficient
, for either
NUP88
NTD
or RNA
binding
(figs. S
47 and S48). To further map
these
interaction
s, we performed
a saturating
NUP358
NTD
surface
mutagenesis
, screening
106 mutants
for NUP88
NTD
and ALPP
SSCR
RNA
binding (
fig. S
49). We found that
positively charged residues
in the concave surface
compositely formed by the N
-terminal solenoid and
the wedge domain
mediate binding
to both
NUP88
NTD
and
RNA. M
utations
that abolished
NUP88
NTD
-binding
clustered
exclusively on the N-
terminal solenoid,
whereas
RNA
disruption
required
additional
mutations in
the wedge domain.
By systematically combining
individual
alanine
substitutions
,
we identified
a NUP358
NTD
2 R5K muta tion ( K34A
, K40A, R58A, K61A, R64A, K500A
, and K502A)
which
abolished both interactions (
fig. S
50).
Next, we
determined the crystal structure of NUP358
OE
a t 1.1 Å
resolution (
table S13
). NUP358
OE
is a small
a
-helical element
that homo-
tetramerizes to
form
an anti
-parallel
four -helix bu
ndle ( Fig. 4
, G
and H, and fig.
S 42A) . The core of the
a
- helical bundle is
lined
with hydrophobic
residues
that co
ordinate
oligomeric
inter -heli cal packing,
demonstrated by
the
monomeric
form assumed by the
NUP358
OE
LIQ IML
mutant
( L811A, I814A,
Q817A
, I821A, M
825A
, a
nd L828A)
(Fig. 4G
and fig. S
42, B
to E). To validate our NUP358
OE
structure, we tested the effect of introducing the LIQIML mutation into
the larger NUP358
NTD- OE
fragment. Whereas wildtype NUP358
NTD- OE
formed higher order oligomeric
species, the oligomerization profile of the LIQIML NUP358
NTD- OE
mutant matched that of t
he OE-
less
NUP358
NTD
, presenting concentration
-dependent dimerization in low salt buffer but persisting in a
monodisperse monomeric state in high salt buffer (
fig. S42 , F and G
).
Our data show that
NUP358
NTD
is composed of three distinct
a
-helical solenoids
that interact
in a
novel manner
, adopting a unique overall
S- shaped architecture
with a propensity to form
15
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint
this version posted October 26, 2021.
;
https://doi.org/10.1101/2021.10.26.465790
doi:
bioRxiv preprint
domain-
swapped
homo-
dimers.
Connected to NUP358
NTD
by a ~50-
residue
linker is
an oligomerization
element
that forms
homo-
tetramers/pe
ntamers
in solution.
These dual modes of homo
-oligomerization
provide a p
lausible
explanation
for NUP358’s
propensity to form phase separation
, as observed during
NPC assembly in
Drosophila
melanogaster
oocytes
(
83
).
Ran interactions
with
human asymmetric nups
Nucleocytoplasmic transport depends on Karyopherin transport factors (Kaps) and a gradient in the state
of the small GTPase Ran from the Ran(GDP)
-high cytoplasm to the Ran(GTP)
-high nucleus
(
2, 7, 8, 10
) .
Import Kap•cargo complexes assembled in the cytoplasm enter the central transp
ort channel of
the NPC
and are disassembled upon arrival in the nucleus by Kap binding to Ran(GTP), which triggers the release
of the bound cargo. Conversely, Ran(GTP) is an obligate component of nuclear export Kap•cargo
complexes, which are disassembled u
pon activation of Ran’s GTPase activity at the cytoplasmic face of
the NPC
. Multiple Ran binding sites are distributed among t
he asymmetric nups at the cytoplasmic and
nuclear
sides
of the NPC
in the form of
distinct
Ran- binding domains (
RanBD
s) and Zn
2+
-finger
(ZnF)
modules
. On the cytoplasmic face
, NUP358
contains
four dispersed
Ran- binding domains (RanBDs)
and
a central zinc finger domain
(ZFD)
with a tandem array of eight ZnFs
( Fig. 4A) (
84
) . On the nuclear side,
NUP153
and NUP50
contain a central ZFD
with four ZnFs
and a solitary C-
terminal R
anBD, respectively
(fig. S51 ) (
85, 86
).
Testing
the Ran(GDP/GTP)-
binding activity
of all 1
7 domains
by SEC-
MALS, we
confirmed
that
all domains bound to
Ran as expected
except for NUP358
ZnF1
( Fig. 4, I and J, and figs.
S51 to S55) .
Consistent with previous reports
, the RanBDs of NUP358
and NUP50
only bound
Ran( GTP)
, whereas
the ZnFs in NUP358
and NUP153
bound
Ran in
both nucleotide states
but showed
a preference for
Ran(GDP)
(figs. S52 and S54) (
84, 87- 89
). To clarify the molecular basis for the different
ial binding
behaviors, we
determined the
co- crystal structures
of all 16 domains bound
to Ran in their
preferred
nucleotide
-bound
state at 1.8 Å-
2.45 Å
resolutions (
tables
S14 to S16
and figs.
S 51 and S55).
All eleven
NUP358
/NUP153
ZnFs
form
small
, ~36-
residue
spherical
domains
stabilized by
a
central
Zn
2+
ion coordinated
by the sulfhydryl
groups of
four conserved cysteines (
Fig. 4K
and fig.
S51 ).
16
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint
this version posted October 26, 2021.
;
https://doi.org/10.1101/2021.10.26.465790
doi:
bioRxiv preprint