Structural and functional analysis of mRNA export regulation by the nuclear pore complex

ARTICLE

Structural and functional analysis of mRNA export

regulation by the nuclear pore complex

Daniel H. Lin

, Ana R. Correia

, Sarah W. Cai

, Ferdinand M. Huber

, Claudia A. Jette

& André Hoelz

The nuclear pore complex (NPC) controls the passage of macromolecules between the

nucleus and cytoplasm, but how the NPC directly participates in macromolecular transport

remains poorly understood. In the

nal step of mRNA export, the DEAD-box helicase DDX19

is activated by the nucleoporins Gle1, Nup214, and Nup42 to remove Nxf1

•

Nxt1 from mRNAs.

Here, we report crystal structures of Gle1

•

Nup42 from three organisms that reveal an evo-

lutionarily conserved binding mode. Biochemical reconstitution of the DDX19 ATPase cycle

establishes that human DDX19 activation does not require IP

, unlike its fungal homologs,

and that Gle1 stability affects DDX19 activation. Mutations linked to motor neuron diseases

cause decreased Gle1 thermostability, implicating nucleoporin misfolding as a disease

determinant. Crystal structures of human Gle1

•

Nup42

•

DDX19 reveal the structural rear-

rangements in DDX19 from an auto-inhibited to an RNA-binding competent state. Together,

our results provide the foundation for further mechanistic analyses of mRNA export in

humans.

DOI: 10.1038/s41467-018-04459-3

OPEN

Division of Chemistry and Chemical Engineering, California Institute of Technology, 1200 East California Boulevard, Pasadena, CA 91125, USA. Thes

authors contributed equally: Ana R. Correia, Sarah W. Cai. Correspondence and requests for materials should be addressed to

A.H. (email:

hoelz@caltech.edu

)

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1

1234567890():,;

T

fl

ow of genetic information requires newly transcribed

and processed mRNAs to be exported from the nucleus to

the cytoplasm through nuclear pore complexes (NPCs).

NPCs are massive macromolecular machines perforating the

nuclear envelope, each composed of ~1000 protein subunits

(collectively termed nucleoporins) totaling to a molecular mass of

~120 MDa in humans

. By fusing the inner and outer nuclear

membranes, NPCs create pores through the nuclear envelope and

simultaneously generate a passive diffusion barrier composed of

disordered protein sequences enriched in phenylalanine-glycine

(FG) repeats. Each NPC is composed of a ~60 MDa symmetric

core that is decorated by different proteins on its nuclear and

cytoplasmic faces, which are referred to as the nuclear basket and

cytoplasmic

fi

lament nucleoporins, respectively (Fig.

a).

The architecture of the symmetric core of the human NPC has

recently been elucidated

. In humans, the symmetric core is

composed of an inner ring that resides in the plane of the

membrane and two concentric outer rings that reside above the

inner and outer nuclear membranes. The outer rings, which serve

as the attachment sites for the nuclear basket and cytoplasmic

Nuclear

basket

Cytoplasmic

filaments

Cytoplasm

Nucleus

Diffusion barrier

(FG repeats)

Symmetric nups

Coat nup complex

Inner ring complex

Nup155

POMs

Asymmetric nups

Cytoplasmic

filaments

Nuclear basket

Nuclear

envelope

Nup155

mutant

E1146A

K1147A

Nup98

binding

Gle1

binding

L1182A

Y1189A

F1192A

I1206A

–

Nup98

Gle1

Both

Neither

Mutation disrupts:

ctNup155

ctNup98

Gle1:

110

Mutation effect:

None

Moderate

No binding

Unassigned

density

Gle1

binding

site

Nup155

Inner

CNC

Outer

CNC

Nup214

450

2090

694

955

-propeller

880

732

498

213

157

APD

Nup98

11391

516

-helical

Nup155

-propeller

DDX19

1479

298

NTD

CTD

1423

379

Nup42

RecA-like domain

Gle1-binding motif

Zinc finger

Auto-inhibitory helix

N-terminal region

Unstructured

FG repeat region

Gle2-binding sequence

Autoproteolytic domain

Coiled-coil domain

Gle1

1698

382

CTD

123 355

Nup155

kDa

Gle1

Nup155

1x Nup98

Gle1

Nup155

3x Nup98

Gle1

Nup155

CTD

•SUMO-Gle1

Nup155

CTD

•SUMO-Gle1

Nup155

CTD

•SUMO-Gle1

+ 1x Nup98

Nup155

CTD

•SUMO-Gle1

+ 3x Nup98

+ 1x Nup98

+ 3x Nup98

1.0

0.8

0.6

0.4

0.2

Absorbance (AU)

Volume (ml)

Fig. 1

Gle1 is anchored to the nuclear pore complex through a competitive interaction with Nup98.

Cartoon schematic of the human nuclear pore

complex (NPC). The circle highlights the region of the NPC to which the proteins used in this study localize.

Domain schematics for nucleoporins used in

this study. Protein names and boundaries correspond to the human proteins.

Size exclusion chromatography (SEC) analysis of the interaction between

Nup155

CTD

, SUMO-Gle1

, and Nup98

. Puri

ed Nup155

CTD

•

SUMO-Gle1

complex was mixed with the indicated amounts of Nup98

and loaded

onto a Superdex 200 10/300 GL column. The gray bar indicates the fractions visualized with Coomassie-stained SDS-PAGE gels.

Left: table summarizing

SEC analysis of Nup155

CTD

variants for Nup98

and SUMO-Gle1

binding. See also Supplementary Fig.

. Right: the homologous positions were colored

on the

thermophilum

Nup170

•

Nup145N structure (

PDB ID 5HB0

), indicating that the same binding surface is recognized by Nup98

and SUMO-Gle1

[

Summary of the effect of Gle1

alanine substitution variants on Nup155

CTD

binding. Colored dots above the sequence of Gle1

indicate the effect of

the substitution, green for wild-type levels of complex formation, orange for reduced binding, or red for complete disruption. See also Supplementa

ry Fig.

Identi

cation of the Gle1 binding site suggests that the unassigned cytoplasmic density adjacent to bridging Nup155 molecules could contain Gle1 and its

binding partners. Left: surface representation of the composite structure of the NPC

. Right: zoomed view of unassigned cytoplasmic density, with

Nup155 shown in orange, Gle1

binding site colored in green, and coat nucleoporin complexes shown in yellow

ARTICLE

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fi

laments, are structurally connected to the inner ring via bridging

Nup155 molecules. In addition to large interaction interfaces

between scaffold proteins, the assembly of the symmetric core

requires interactions mediated by

fl

exible linker sequences

residing in Nup98, Nup53, and Nup93

. In contrast, the

molecular organization of the cytoplasmic

fi

laments and nuclear

basket remains poorly understood.

Preparation of mRNAs for nuclear export is a highly coordi-

nated process that begins co-transcriptionally and results in the

addition and removal of mRNA-binding proteins during tran-

scription and nuclear processing until an export-competent

messenger ribonucleoprotein particle (mRNP) is formed

Although some mRNAs may be exported through specialized

pathways, the bulk of mRNA export is mediated by the evolu-

tionarily conserved, heterodimeric transport factor complex

Nxf1

•

Nxt1

. Nxf1

•

Nxt1 binds mRNAs without strong sequence

preference and can shuttle mRNAs through the NPC diffusion

barrier by binding to FG repeats. At the cytoplasmic face of the

NPC, Nxf1

•

Nxt1-bound mRNPs encounter the cytoplasmic

fi

la-

ment nucleoporins Gle1, Nup42, and Nup214, which speci

fi

cally

activate the DEAD-box helicase DDX19 to remove Nxf1

•

Nxt1

from the mRNP

. This spatial regulation of activity prevents re-

import of mRNPs into the nucleus, and thus ensures the direc-

tionality of mRNA export.

DDX19 is a member of the DEAD-box helicase family, which

are RNA-dependent ATPases composed of two RecA domains

(referred to as DDX19

NTD

and DDX19

CTD

throughout the text)

(Fig.

b). Many insights into the regulation of DDX19 come from

studies of the fungal homolog, Dbp5. Genetic, biochemical, and

structural studies of the yeast proteins have revealed that the

interaction between Gle1 and Dbp5 occurs via their C-terminal

domains (CTDs) and is bridged by the small molecule inositol

hexaphosphate (IP

)

–

. In yeast, Gle1, IP

, and RNA cooperate

to stimulate the ATPase activity of Dbp5, although the precise

mechanism remains debated

. The functional roles of Nup42

and Nup214 are also unclear. The interaction between Nup214

and DDX19 is required for steady-state localization of DDX19 to

the NPC, but Nup214 binding also inhibits DDX19 activity

–

Nup42 binds to Gle1, but its contributions to DDX19 activity

are unknown

. DDX19 and Gle1 have been implicated in

other cellular processes including transcription regulation,

DNA damage response, translation initiation, and RNA

processing

–

Impairment of nucleocytoplasmic transport through the NPC

has been linked to both Huntington

’

s disease and amyotrophic

lateral sclerosis (ALS)

–

. Nucleocytoplasmic transport factors

and nucleoporins, including Gle1, are genetic modi

fi

ers of disease

in model systems and are mislocalized in both model organism

and patient samples

–

. Speci

fi

c mutations of Gle1 are

associated with lethal contracture congenital syndrome 1

(LCCS1), lethal arthrogryposis with anterior horn cell disease

(LAAHD), and ALS

. However, deciphering how defects in

nucleocytoplasmic transport can lead to disease will require an

improved understanding of the regulation of transport through

the NPC.

To gain further mechanistic insight into the role of nucleo-

porins in mRNA export, we characterized the molecular archi-

tecture of the cytoplasmic

fi

lament nucleoporins involved in

regulating DDX19 activity. We mapped the Gle1-binding site on

Nup155 and found that it overlaps with the Nup98 binding site,

thereby acquiring a spatial restraint for Gle1 localization in the

NPC. Crystal structures of the Gle1

•

Nup42 complex from

Sac-

charomyces cerevisiae

Chaetomium thermophilum

, and

Homo

sapiens

revealed the evolutionarily conserved structural basis of

their interaction. Nup42 is critical for the thermostability of

human Gle1, enabling Gle1 puri

fi

cation from a recombinant

source and characterization of its role in human DDX19 activa-

tion, which unlike the yeast system does not require IP

binding.

Crystal structures of the human Gle1

•

Nup42

•

DDX19 complex

bound to ADP and AMP-PNP

•

+

uncovered the adaptations

that facilitate IP

-independent activation in humans and the

speci

fi

c Gle1-induced conformational changes that release

DDX19 from an auto-inhibited state. Lastly, Gle1 mutations that

are associated with motor neuron diseases possess severe ther-

mostability defects, suggesting that nucleoporin misfolding con-

tributes to disease.

Results

Gle1 and Nup98 recognize overlapping surfaces on Nup155

.To

gain a better understanding of the molecular architecture of the

nucleoporins that regulate mRNA export, we set out to recon-

stitute the interactions with puri

fi

ed, recombinant proteins. We

use the names of the human proteins unless otherwise speci

fi

ed.

In humans, there are two splice variants of the Gle1 transcript,

Gle1A and Gle1B, which encode proteins that are primarily

localized in the cytoplasm or at the cytoplasmic face of the NPC,

respectively

. We focused on the NPC-localized Gle1B, referred

to as Gle1 throughout the text. Human Gle1 can be divided into

three structural domains: an unstructured N-terminal region

(Gle1

, residues 1

−

123), a coiled-coil region (Gle1

, residues

124

–

355), and a highly conserved CTD (Gle1

CTD

, residues

382

–

698) (Fig.

b). Gle1

CTD

is the domain that binds and sti-

mulates DDX19, but previous studies have identi

fi

ed features in

all three regions that are important for NPC localization

We began our analysis by focusing on the interaction between

Gle1 and the adaptor nucleoporin Nup155 (Nup170 in fungi).

Although the

fi

rst 28 residues of Gle1 were previously shown to

be suf

fi

cient for an interaction, we used a SUMO-fusion construct

that also included several charged residues at the C-terminus to

enhance protein solubility (SUMO-Gle1

, residues 2

–

33)

. The

Nup155 CTD (Nup155

CTD

, residues 870

–

1391) and SUMO-

Gle1

formed a stoichiometric complex in size exclusion

chromatography (SEC) experiments (Fig.

c; Supplementary

Fig.

a). Nup155

CTD

also contains a binding site for Nup98,

which is both a component of the symmetric core of the NPC and

of the cytoplasmic

fi

laments

. Stoichiometric complex formation

also occurred between Nup155

CTD

and a construct of Nup98

lacking the FG repeat region (Nup98

Δ

, residues 498

–

880)

(Supplementary Fig.

b). However, when we attempted to

reconstitute a heterotrimeric complex by adding Nup98

Δ

Nup155

CTD

•

SUMO-Gle1

, complex formation between

Nup155

CTD

and Nup98

Δ

coincided with displacement of

SUMO-Gle1

, suggesting that the interactions were mutually

exclusive (Fig.

c; Supplementary Fig.

a). Similarly, addition of

SUMO-Gle1

to Nup155

CTD

•

Nup98

Δ

failed to yield a

heterotrimeric complex (Supplementary Fig.

b). This effect was

speci

fi

c to Nup98, as addition of the N-terminal domain of

Nup214 (Nup214

NTD

) to Nup155

CTD

•

SUMO-Gle1

did not

displace SUMO-Gle1

(Supplementary Fig.

c). In GST-pull-

down experiments, Gle1

fi

ciently outcompeted Nup98

Δ

for

Nup155

CTD

binding, whereas conversely, Nup98

Δ

could not

outcompete Gle1

, suggesting that Gle1

binds with greater

fi

nity to Nup155

CTD

than Nup98

Δ

(Supplementary Fig.

d).

We next expanded upon previous mutational and structural

analyses using the

C. thermophilum

proteins

. Amino acid

substitutions at positions homologous to those that abolished

binding for the

C. thermophilum

proteins also disrupted binding

between human Nup155

CTD

and Nup98

Δ

, demonstrating that

the mechanism of interaction between these two nucleoporins is

evolutionarily conserved (Fig.

d; Supplementary Fig.

a). More-

over, most residues that were critical for Nup98

Δ

binding were

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3

also important for Gle1

binding, except for two residues that

each affected only one nucleoporin, indicating that Gle1

and

Nup98

Δ

recognize overlapping surfaces on Nup155

CTD

(Fig.

Supplementary Fig.

b). Hydrophobic residues in Gle1

were

critical for Nup155

CTD

binding, consistent with utilization of the

same hydrophobic pockets as Nup98

Δ

(Fig.

e; Supplementary

Fig.

c).

In summary, we found that Gle1

and Nup98

Δ

recognize

overlapping surfaces on Nup155

CTD

. Nup155 molecules bridge

the inner ring to the cytoplasmic and nuclear outer coat

nucleoporin complex (CNC) double rings (Fig.

. Because

Gle1

fi

ciently outcompetes Nup98

Δ

for binding, Gle1 would

likely prevail for binding to Nup155 molecules exposed to the

cytoplasmic face. Indeed, in the cytoplasmic outer ring, but not

the nuclear outer ring, there is a volume of unaccounted density

directly adjacent to the Nup155

CTD

surface that binds Gle1

(Fig.

f). Our analysis suggests this density contains the

remainder of the Gle1 molecule and its binding partners.

Identi

fi

cation of a minimal Nup42 Gle1-binding fragment

.We

next focused on the interaction between Gle1

CTD

and Nup42.

Previous studies have shown that the C-terminal, non-FG repeat

region of Nup42 binds Gle1

CTD

. To identify the minimally

suf

fi

cient fragment of Nup42 that recognizes Gle1, we monitored

the localization of Gle1-GFP and mCherry-HA-tagged Nup42

variants in

cerevisiae

. Nup42 truncations that contained

residues 397

–

430 displayed nuclear rim staining consistent with

localization to the NPC, whereas an Nup42 variant containing

only residues 410

–

430 did not (Fig.

a). From these results, we

concluded that the Nup42 Gle1-binding motif (Nup42

GBM

)is

located within residues 397

–

430 (See Supplementary Note

When we recombinantly puri

fi

ed the minimal

S. cerevisiae

Gle1

CTD

•

Nup42

GBM

complex, we noticed that the puri

fi

complex was more stable than

apo

Gle1

CTD

, which typically

requires the addition of IP

in puri

fi

cation buffers for stability

This observation led us to test the effect of IP

and Nup42

GBM

the stability of Gle1

CTD

using two different assays: differential

scanning

fl

uorimetry and a protein solubility assay. In both

experiments, IP

potently improved Gle1

CTD

stability, with

saturating amounts of IP

shifting the melting temperature

(

) from 22 to 37 °C (Fig.

b; Supplementary Fig.

a).

Nup42

GBM

had an even more dramatic effect, increasing the

from 22 to 46 °C, and the presence of both IP

and Nup42

GBM

shifted the

of Gle1

CTD

to 53 °C (Fig.

b; Supplementary

Fig.

a).

The dif

fi

culty of purifying human Gle1

CTD

has previously

prevented a detailed biochemical analysis of the human proteins,

but the dramatic effect of yeast Nup42

GBM

on Gle1

CTD

stability

led us to test whether human Nup42

GBM

has the same effect on

human Gle1

CTD

. Co-puri

fi

cation of a homologous human

Gle1

CTD

•

Nup42

GBM

complex (containing Gle1 residues 382

−

698 and Nup42 residues 379

−

423) resulted in dramatic

improvements in the stability of Gle1

CTD

and the yields increased

from ~0.5 to ~50 mg/100 l of bacterial culture. In thermostability

experiments, the

of human Gle1

CTD

increased from 37 to 50 °

C in the presence of Nup42

GBM

(Fig.

c; Supplementary Fig.

b).

However, in contrast to the yeast proteins, IP

had almost no

effect on thermostability for human Gle1

CTD

(Fig.

c; Supple-

mentary Fig.

b). In summary, we identi

fi

ed an evolutionarily

conserved C-terminal fragment of Nup42 that binds Gle1 and has

a profound effect on Gle1 stability in both yeast and humans.

Evolutionary conservation of the Gle1

−

Nup42 interaction

.To

understand the molecular basis for the interaction between

Gle1

CTD

and Nup42

GBM

and the resulting stabilization of

Gle1

CTD

, we determined the crystal structure of

S. cerevisiae

Gle1

CTD

•

Nup42

GBM

at 1.75 Å resolution (Fig.

d; Supplementary

Table

). Nup42

GBM

folds into a compact domain with a

hydrophobic core that buries a solvent-exposed hydrophobic

surface on Gle1

CTD

, yielding an interaction interface area of

~835 Å

(Fig.

d; Supplementary Fig.

a; See Supplementary

Note

for details). Consistent with the extensive interaction

surface, the interaction between Gle1

CTD

and Nup42

GBM

was

robust against several individual alanine substitutions in SEC

experiments (Supplementary Fig.

a). Instead, highly disruptive

substitutions that introduced negative charge into the hydro-

phobic core (F414D or F409D/F414D) were required to com-

pletely disrupt the interaction between Gle1

CTD

and Nup42

GBM

(Supplementary Fig.

b). In agreement with these results, Nup42

variants containing the F414D or F409D/F414D substitutions

were unable to rescue Nup42 deletion in

S. cerevisiae

(Supple-

mentary Fig.

). In contrast, the F409D substitution in the Nup42

hydrophobic core did not ablate binding, but did alter the elution

pro

fi

le, suggesting a conformational difference in the complex

(Supplementary Fig.

b). Accordingly, an Nup42 variant har-

boring the F409D substitution only had a mild effect on growth at

37 °C (Supplementary Fig.

a).

To evaluate whether the mode of interaction observed for

S. cerevisiae

Gle1

CTD

•

Nup42

GBM

was conserved in other

eukaryotes, we determined the crystal structures of human

Gle1

CTD

•

Nup42

GBM

at 2.8 Å resolution and of

C. thermophilum

Gle1

CTD

•

Nup42

GBM

in the presence and absence of IP

at 2.17

and 2.65 Å resolution, respectively (Fig.

e, f; Supplementary

Fig.

a; Supplementary Tables

and

). The overall structure of

Gle1

CTD

is conserved between fungi and humans, with minor

differences resulting from small insertions or different loop sizes

(Fig.

g; Supplementary Fig.

and Supplementary Movie

). Both

human and

C. thermophilum

Nup42

GBM

adopt the same fold as

S. cerevisiae

Nup42

GBM

and recognize the same surface on

Gle1

CTD

, with the critical hydrophobic residues nearly universally

conserved (Fig.

i, j; Supplementary Figs.

–

; See Supplemen-

tary Note

for details). Many of the critical interaction interfaces

in the NPC likely possess a similar degree of structural

conservation. Burial of the exposed hydrophobic residues and

the thermodynamic favorability of Nup42

GBM

binding and

folding explain the large effect Nup42

GBM

has on Gle1

CTD

stability. Nup42

GBM

binding may also help prevent Gle1

CTD

from sampling partially unfolded states that could lead to

aggregation. The remainder of Nup42 is comprised primarily of

FG repeats. In yeast, deletion of these FG repeats was detrimental

when combined with the deletion of other FG repeats in the

cytoplasmic

fi

laments

. Thus, in addition to ensuring the

stability of Gle1

CTD

, Nup42

GBM

also has a role in anchoring

FG repeats proximal to Gle1 in the NPC.

The IP

binding pocket is not conserved in metazoan Gle1

.In

yeast, activation of Dbp5, the fungal homolog of DDX19, requires

the small molecule IP

, which binds to a highly positively charged

pocket in Gle1

CTD

adjacent to the Dbp5 binding surface and

bridges the two proteins

–

. Our Gle1

CTD

•

Nup42

GBM

structure

reveals that Nup42

GBM

interacts with Gle1

CTD

at a surface that is

distinct and well separated from the IP

binding pocket and the

Dbp5 interface (Supplementary Fig.

. Thus, in order for

Nup42

GBM

binding to affect Dbp5 activation, it would have to do

so through an allosteric mechanism. However, there were mini-

mal conformational differences in Gle1

CTD

between our

Gle1

CTD

•

Nup42

GBM

structure and the previously reported

structure of Gle1

CTD

•

Dbp5 (Supplementary Fig.

Similarly,

C. thermophilum

Gle1

CTD

•

Nup42

GBM

undergoes

minimal conformational changes upon IP

binding, which are

mostly limited to a loop directly adjacent to the IP

pocket. IP

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binds to

C. thermophilum

Gle1

CTD

and to

S. cerevisiae

Gle1

CTD

in a similar orientation, suggesting that IP

could function

similarly in

C. thermophilum

as in

S. cerevisiae

(Fig.

a, b, d, e;

Supplementary Fig.

d; See Supplementary Note

). However,

because there are no

apo

Gle1

CTD

structures available, we could

not exclude the possibility that Nup42

GBM

binding caused

Gle1

CTD

to adopt the same conformation as the one observed

upon IP

and Dbp5 binding.

scGle1

CTD

+ IP

scGle1

CTD

•Nup42

GBM

+ IP

hsGle1

CTD

+ IP

hsGle1

CTD

•Nup42

GBM

+ IP

Gle1

Nup42

Gle1

Nup42

180°

scGle1

CTD

•Nup42

GBM

hsGle1

CTD

•Nup42

GBM

ctGle1

CTD

•Nup42

GBM

scGle1

CTD

•Nup42

GBM

hsGle1

CTD

•Nup42

GBM

ctGle1

CTD

•Nup42

GBM

Superposition

Gle1

Nup42

DIC

Nup42-

mCherry-HA

Gle1-

GFP

DIC

Nup42-

mCherry-HA

Gle1-

GFP

30 °C

42 °C, 3 h

Gle1

Nup42

Extended

N-terminus

nup42

/gle1-GFP

430

397

mCherry

Nup42

364-430

Nup42

397-430

Nup42

410-430

Nup42

1-394

F409

P424

P425

P423

Y488

W451

L495

Q491

L401 L428

D421

V419

R456

I408

K494

F414

P420

N459

A455

L498

P426

P417

P416

P415

I411

Y637

Q640

I385

W602

M644

L413

N610

L647

P412

K643

F401

F406

F539

Y521 V520

P548

P547

P544

Y484

W447

F533

W530

N455

A451

M543

A491

100

Temperature (°C)

100

Temperature (°C)

RFU (×10

)

RFU (×10

)

Fig. 2

A conserved mechanism for Gle1

•

Nup42 complex formation.

In vivo localization analysis of Gle1-GFP and Nup42-mCherry-HA variants in

nup42

Δ

gle1-GFP S. cerevisiae

cells. Scale bar is 5

μ

m. Schematics on the right indicate the Nup42 fragments that were included in the construct, with omitted

fragments indicated by replacement of the domain with a solid line. Residue numbers indicate the fragment included in each construct. See also

Supplementary Fig.

Differential scanning

uorimetry analysis of

S. cerevisiae

H. sapiens

Gle1

CTD

thermostability in the presence and absence of

Nup42

GBM

and IP

. Exposure of hydrophobic residues was monitored by an increase in relative

uorescence units (RFUs). Curves represent the average of

three experiments. See also Supplementary Fig.

−

Crystal structures of

S. cerevisiae

H. sapiens

,or

C. thermophilum

Gle1

CTD

•

Nup42

GBM

. See also

Supplementary Figs.

and

–

Superposition of the structures of

S. cerevisiae, H. sapiens

, and

C. thermophilum

Gle1

CTD

•

Nup42

GBM

, with same coloring as

−

. See also Supplementary Movie

−

Zoom views of

S. cerevisiae

H. sapiens

,or

C. thermophilum

Gle1

CTD

•

Nup42

GBM

interactions with residues

mediating the interaction labeled

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5

In contrast to the IP

binding pockets of

S. cerevisiae

and

thermophilum

Gle1

CTD

, the same location on the surface of the

H. sapiens

molecule was signi

fi

cantly altered, resulting in a

substantial reduction in positive electrostatic potential (Fig.

−

f). This was the consequence of several positively charged

residues that are essentially invariant in fungi (

S. cerevisiae

residues K264, K286, K333, and R374;

C. thermophilum

residues

K225, R249, K327, and K374), but are not conserved in humans

(V401, Q423, E482, and H523) (Fig.

−

c). This trend held for all

the metazoan sequences inspected, indicating that the altered

abc

def

scGle1

CTD

•Nup42

GBM

/ Dbp5 binding pocket

ctGle1

CTD

•Nup42

GBM

/ Dbp5 binding pocket

hsGle1

CTD

•Nup42

GBM

DDX19 binding pocket

K257

K378

K286

W326

K264

H337

E340

K333

R374

N329

K272

V268

I260

K401

K377

H221

K374

K327

K229

Y320

R249

R253

R228

K225

R222

W401

I330

K526

T408

Q423

T427

K479

K486

E482

E489

K527

Q394

M398

S397

V401

H523

Y478

scDbp5

kDa

scDbp5

scGle1

kDa

ctGle1

kDa

Gle1

kDa

ctDbp5

kDa

ctDbp5

ctGle1

kDa

scDbp5

scGle1

kDa

ctDbp5

ctGle1

DDX19

kDa

DDX19

Gle1

kDa

DDX19

Gle1

Dbp5 + Gle1

CTD

•Nup42

GBM

no IP

Dbp5 + Gle1

CTD

•Nup42

GBM

+ IP

Dbp5

Gle1

CTD

•Nup42

GBM

+ IP

Gle1

CTD

•Nup42

GBM

+ IP

Gle1

CTD

•Nup42

GBM

no IP

Dbp5 + Gle1

CTD

•Nup42

GBM

no IP

Dbp5 + Gle1

CTD

•Nup42

GBM

+ IP

Gle1

CTD

•Nup42

GBM

no IP

Dbp5

Gle1

CTD

•Nup42

GBM

+ IP

Dbp5 + Gle1

CTD

•Nup42

GBM

no IP

Dbp5 + Gle1

CTD

•Nup42

GBM

+ IP

Gle1

CTD

•Nup42

GBM

no IP

DDX19

Gle1

CTD

•Nup42

GBM

+ IP

DDX19 + Gle1

CTD

•Nup42

GBM

no IP

DDX19 + Gle1

CTD

•Nup42

GBM

+ IP

Gle1

CTD

•Nup42

GBM

no IP

Dbp5 + Gle1

CTD

•Nup42

GBM

no IP

Dbp5 + Gle1

CTD

•Nup42

GBM

+ IP

Dbp5

Gle1

CTD

•Nup42

GBM

+ IP

Gle1

CTD

•Nup42

GBM

no IP

DDX19 + Gle1

CTD

•Nup42

GBM

no IP

DDX19 + Gle1

CTD

•Nup42

GBM

+ IP

DDX19

Gle1

CTD

•Nup42

GBM

+ IP

Gle1

CTD

•Nup42

GBM

no IP

S. cerevisiae

C. thermophilum

–10 kT/e

+10 kT/e

–10 kT/e

+10 kT/e

–10 kT/e

+10 kT/e

scGle1

kDa

ctGle1

kDa

Gle1

kDa

1.2

1.0

0.8

0.6

0.4

0.2

Absorbance (AU)

Volume (ml)

1.2

1.0

0.8

0.6

0.4

0.2

Volume (ml)

H. sapiens

1.2

1.0

0.8

0.6

0.4

0.2

Volume (ml)

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6

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electrostatic potential in this pocket may be a general feature of

metazoan Gle1 (Supplementary Fig.

a). Although there are two

positions in human Gle1 that contained lysine residues not

present in the fungal proteins, sequence conservation analysis also

indicated that the positively charged residues in Dbp5 that bind

were not conserved in metazoans (Supplementary Fig.

b).

Combined, these structural observations raised the question of

what roles Nup42 and IP

had on the activation of human

DDX19.

The roles of IP

and Nup42 in DDX19/Dbp5 stimulation

.To

assess the role of IP

in DDX19 activation, we

fi

rst tested whether

complex formation between human DDX19 and Gle1 was

dependent on IP

in SEC experiments. Consistent with previous

reports,

S. cerevisiae

Dbp5 and Gle1

CTD

•

Nup42

GBM

only formed

a complex in the presence of IP

(Fig.

. The interaction

between

C. thermophilum

Gle1

CTD

•

Nup42

GBM

and Dbp5 also

required IP

, consistent with the evolutionary conservation of the

binding residues among fungi (Fig.

h,; Supplementary

Fig.

a). In contrast, complex formation of the human proteins

did not require IP

, and the presence of IP

instead partially

reduced complex formation (Fig.

i).

To directly test the effect of IP

and Nup42 on Dbp5 and

DDX19 activity, we measured steady-state ATP hydrolysis rates

with an NADH-coupled reaction using conditions identical to

those previously reported

. Due to concerns that observed

differences in activity were due to the stabilizing effects of IP

and

Nup42 on Gle1 rather than direct roles in Dbp5/DDX19

activation, we measured ATP hydrolysis rates at multiple

temperatures to decouple the effects due to stabilization from

bona

fi

de stimulatory roles. Because human DDX19 was

intrinsically less active than

S. cerevisiae

Dbp5, we used

fi

vefold

higher concentrations of human DDX19, Gle1

CTD

, Nup42

GBM

and IP

to ensure accurate measurements of ATPase activity.

Furthermore, because Dbp5 and DDX19 are slow ATPases, we

developed extensive protein puri

fi

cation protocols to ensure all of

the measured activity was directly attributable to Dbp5 or DDX19

(Supplementary Fig.

a, b).

In the yeast system, and across a range of RNA concentrations,

the addition of Nup42

GBM

to Gle1

CTD

increased Dbp5 activity at

37 °C, but this stimulatory effect was greatly diminished at 30 °C,

a temperature below the

of Gle1

CTD

•

, which is consistent

with thermostability having a detectable effect in our assays

(Fig.

a, c; Supplementary Fig.

c). Altogether, these data

indicate that Nup42

GBM

does not have a direct role in

Dbp5 stimulation in the yeast system, but rather functions

primarily to ensure Gle1 stability. In the yeast system, IP

was

required for full stimulation of Dbp5 at all temperatures tested,

indicating that in addition to its ability to stabilize yeast Gle1

CTD

also has a direct role in Dbp5 stimulation (Fig.

Supplementary Fig.

c, d). These results are in agreement with

the observation that stable association of Gle1

CTD

and Dbp5

requires IP

binding. In contrast, neither Nup42

GBM

nor IP

were

stimulatory in the human system at either temperature we tested

(Fig.

b, d; Supplementary Fig.

e).

Because differences in the IP

binding pocket in human Gle1

and DDX19 could have weakened the af

fi

nity for IP

rather than

completely abolishing binding, we tested whether higher

concentrations of IP

would stimulate DDX19. IP

stimulated

Dbp5 activity in a dose-dependent manner that saturated at 1

(twice the Dbp5 concentration), but had no effect on human

DDX19 activity up to concentrations of 100

M, which is an

upper estimate for the total (bound and free) IP

concentration in

human cells (Fig.

. Additionally, IP

bound tightly to

cerevisiae

Gle1

CTD

•

Nup42

GBM

in isothermal titration calorimetry

experiments (ITC) with a dissociation constant (

) of ~3 nM.

However, no binding of IP

to human Gle1

CTD

•

Nup42

GBM

was

detected up to concentrations of 200

M (Supplementary Fig.

Taken together, our results indicate that IP

binding is conserved

in fungi but not in humans. Moreover, sequence analysis of other

metazoan Gle1 and DDX19 sequences suggests that IP

binding

may not be a feature of metazoan DDX19 activation in general, as

-binding residues are not present in a diverse array of

metazoan sequences (Supplementary Fig.

a). These results do

not exclude the possibility that another small molecule may serve

a similar function in humans as IP

does in fungi. However,

unlike their fungal homologs, the human proteins alone are

suf

fi

cient for complex formation.

Structural basis for IP

-independent human DDX19 activa-

tion

. To understand the structural basis for IP

-independent

DDX19 activation, we determined crystal structures of human

Gle1

CTD

•

Nup42

GBM

in complex with DDX19 in the presence of

ADP or AMP-PNP

•

+

at 3.6 and 3.4 Å resolution, respectively

(Fig.

a, d; Supplementary Table

). Previous structural studies

have revealed that DDX19 possesses an auto-inhibitory helix N-

terminal to DDX19

NTD

(residues 54

–

67) that can bind between

DDX19

NTD

and DDX19

CTD

, preventing RNA binding or for-

mation of a catalytically competent active site

. This helix

appears to be conserved among metazoan DDX19 sequences, but

is absent in their fungal Dbp5 homologs (Supplementary Fig.

Therefore, we used a construct of DDX19 that contained the N-

terminal auto-inhibitory helix but did not contain the

fl

exible N-

terminal extension (DDX19

Δ

N53

, residues 54

–

479). In addition,

we determined the crystal structure of

apo

DDX19

Δ

N53

(AMP-

PNP

•

+

) at 2.2 Å resolution (Supplementary Fig.

a; Sup-

plementary Table

The heterotrimeric Gle1

CTD

•

Nup42

GBM

•

DDX19

Δ

N53

complex

exhibited similar conformations in the presence of ADP or AMP-

PNP

•

+

, with the auto-inhibitory helix still bound between

DDX19

NTD

and DDX19

CTD

(Fig.

a, d). The surprising

observation that AMP-PNP-bound DDX19 could also adopt the

auto-inhibited conformation was con

fi

rmed by the structure of

apo

DDX19

Δ

N53

(AMP-PNP

•

+

), which adopted an identical

conformation as

apo

DDX19

Δ

N53

(ADP) (Supplementary

Fig.

. The inhibited state does not differ structurally in

the presence of ADP or ATP, as the nucleotide-binding pocket

readily accommodates the additional phosphate and Mg

+

ion,

with minor changes in the sidechain conformations of K64 from

Fig. 3

Human Gle1

CTD

binding to DDX19 is IP

independent.

−

Zoom view of the IP

binding pocket of

S. cerevisiae

C. thermophilum

,or

H. sapiens

Gle1

CTD

. Residues that are conserved in fungi but not metazoans are highlighted in red. See also Supplementary Figs.

and

−

Surface electrostatic

potential analysis of IP

binding pockets for

S. cerevisiae

C. thermophilum

,or

H. sapiens

Gle1. The same view as

−

is shown in surface representation

colored by electrostatic potential, from red (

−

10 k

T/e) to white (0 k

T/e) to blue (

+

10 k

T/e), revealing a dramatically reduced electrostatic potential for

human Gle1

CTD

. See also Supplementary Figs.

, and

−

SEC analysis of the interaction between Dbp5/DDX19 and Gle1

CTD

•

Nup42

GBM

for

cerevisiae

C. thermophilum

,or

H. sapiens

, in the presence or absence of IP

. The elution pro

les for Dbp5/DDX19 are shown in purple,

Gle1

CTD

•

Nup42

GBM

and IP

are shown in gray, Gle1

CTD

•

Nup42

GBM

without IP

are shown in orange, Dbp5/DDX19 with Gle1

CTD

•

Nup42

GBM

and IP

are

shown in light blue, and Dbp5/DDX19 with Gle1

CTD

•

Nup42

GBM

without IP

are shown in black. Fungal Gle1

CTD

•

Nup42

GBM

interacts strongly with the

Superdex matrix in the absence of IP

. The gray horizontal bar indicates fractions visualized with Coomassie-stained SDS-PAGE gels shown below

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7