Comparative structural analysis of human DEAD-box RNA helicases.

Abstract

DEAD-box RNA helicases play various, often critical, roles in all processes where RNAs are involved. Members of this family of proteins are linked to human disease, including cancer and viral infections. DEAD-box proteins contain two conserved domains that both contribute to RNA and ATP binding. Despite recent advances the molecular details of how these enzymes convert chemical energy into RNA remodeling is unknown. We present crystal structures of the isolated DEAD-domains of human DDX2A/eIF4A1, DDX2B/eIF4A2, DDX5, DDX10/DBP4, DDX18/myc-regulated DEAD-box protein, DDX20, DDX47, DDX52/ROK1, and DDX53/CAGE, and of the helicase domains of DDX25 and DDX41. Together with prior knowledge this enables a family-wide comparative structural analysis. We propose a general mechanism for opening of the RNA binding site. This analysis also provides insights into the diversity of DExD/H- proteins, with implications for understanding the functions of individual family members.

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Comparative Structural Analysis of Human DEAD-Box
RNA Helicases
Patrick Schu ¨tz1, Tobias Karlberg1, Susanne van den Berg1, Ruairi Collins1, Lari Lehtio ¨1¤a, Martin
Ho ¨gbom1¤b, Lovisa Holmberg-Schiavone1¤c, Wolfram Tempel2, Hee-Won Park2, Martin Hammarstro ¨m1,
Martin Moche1, Ann-Gerd Thorsell1, Herwig Schu ¨ler1*
1Structural Genomics Consortium, Karolinska Institutet, Stockholm, Sweden, 2Structural Genomics Consortium and Department of Pharmacology, University of Toronto,
Toronto, Canada
Abstract
DEAD-box RNA helicases play various, often critical, roles in all processes where RNAs are involved. Members of this family of
proteins are linked to human disease, including cancer and viral infections. DEAD-box proteins contain two conserved
domains that both contribute to RNA and ATP binding. Despite recent advances the molecular details of how these
enzymes convert chemical energy into RNA remodeling is unknown. We present crystal structures of the isolated DEAD-
domains of human DDX2A/eIF4A1, DDX2B/eIF4A2, DDX5, DDX10/DBP4, DDX18/myc-regulated DEAD-box protein, DDX20,
DDX47, DDX52/ROK1, and DDX53/CAGE, and of the helicase domains of DDX25 and DDX41. Together with prior knowledge
this enables a family-wide comparative structural analysis. We propose a general mechanism for opening of the RNA
binding site. This analysis also provides insights into the diversity of DExD/H- proteins, with implications for understanding
the functions of individual family members.
Citation: Schu ¨tz P, Karlberg T, van den Berg S, Collins R, Lehtio ¨ L, et al. (2010) Comparative Structural Analysis of Human DEAD-Box RNA Helicases. PLoS ONE 5(9):
e12791. doi:10.1371/journal.pone.0012791
Editor: Nick Gay, University of Cambridge, United Kingdom
Received June 17, 2010; Accepted August 14, 2010; Published September 30, 2010
Copyright: ? 2010 Schu ¨tz et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: The Structural Genomics Consortium is a registered charity (number 1097737) that receives funds from the Canadian Institutes for Health Research, the
Canada Foundation for Innovation, Genome Canada through the Ontario Genomics Institute, GlaxoSmithKline, Karolinska Institutet, the Knut and Alice
Wallenberg Foundation, the Ontario Innovation Trust, the Ontario Ministry for Research and Innovation, Merck & Co., Inc., the Novartis Research Foundation, the
Swedish Agency for Innovation Systems, the Swedish Foundation for Strategic Research and the Wellcome Trust. The funders had no role in study design, data
collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: Clarifying statements: 1. The Structural Genomics Consortium (SGC) is a not-for-profit organization that receives funding from a funder
consortium that includes commercial sources (GlaxoSmithKline and Merck & Co., Inc.). This circumstance does not alter the authors’ adherence to all the PLoS ONE
policies on sharing data and materials. The SGC and its scientists are committed to making their research outputs (materials and knowledge) available without
restriction on use. This means that the SGC will promptly place its results in the public domain and will not agree to file for patent protection on any of its
research outputs. It will seek the same commitment from any research collaborator. 2. One of the authors (LHS) is currently employed by a commercial company.
As the role of this author in the current study was terminated before her affiliation with that company, this circumstance does not alter the authors’ adherence to
all the PLoS ONE policies on sharing data and materials.
* E-mail: herwig.schuler@ki.se
¤a Current address: Department of Biosciences, A˚bo Akademi, Turku, Finland
¤b Current address: Department of Biochemistry and Biophysics, Stockholm University, Stockholm, Sweden
¤c Current address: Structural Chemistry Laboratory, AstraZeneca R&D, Mo ¨lndal, Sweden
Introduction
DExD/H-box RNA helicases, from virus and bacteria to
eukaryotes, play important roles in processes including ribosome
biogenesis, RNA processing and folding, ribonucleoprotein (RNP)
remodeling, RNA nuclear export, the regulation of RNA
translation and transcription, and nonsense-mediated RNA decay.
DExD/H-box RNA helicases have multiple functions in these
processes: They can act as RNA chaperones, ATP-dependent
RNA helicases and unwindases, as RNPases by mediating RNA-
protein association and dissociation [1–4] or as co-activators and
co-repressors of transcription ([5–7] and refs. therein). Cancer cell
lines often feature deregulated expression or impaired functioning
of RNA helicases [5,8]. In addition, several family members are
captured and regulated by viral proteins [9], are involved in viral
RNA maturation [10], or mediate antiviral host defense [11,12].
Inhibition of individual RNA helicases as a therapeutic route is
currently being explored (e.g., [13–17]).
DExD/H-box proteins often contain accessory regulatory
domains and localization modules, but their cores consist of two
RecA-like domains joined by a short flexible linker. The N-
terminal domain is commonly referred to as conserved domain-1,
or DEAD-domain, and the C-terminal domain as conserved
domain-2, or helicase domain [3,4,18]. Both domains contribute
to the binding site for RNA substrates and both contribute to ATP
hydrolysis. These activities are coupled to one another by allostery
throughout the protein molecules. Consequently, a detailed
understanding of how these proteins convert chemical energy into
RNA remodeling requires knowledge of the structures of the two
conserved domains independent of each other and interacting in
the closed active state. To date, crystal structures of tandem
domains are available for several DExD/H-box helicases, also in
complex with RNA substrates [19–22]. To understand the RNA
remodeling event and the underlying structural rearrangements, it
is important to compare these structures with those of each
domain in isolation.
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We have solved crystal structures of single domains from eleven
human DExD/H-box helicases of the DEAD-motif subfamily. A
comparative analysis of these structures uncovered not only
isoform specific features, but also nucleotide specific positioning of
flexible elements that are common to several proteins. We suggest
a structural mechanism for the linkage between binding of ATP
and activation of the RNA binding site.
Results and Discussion
We used X-ray crystallography to determine the structures of the
DEAD-domains of DDX2A, DDX2B, DDX5, DDX10, DDX18,
DDX20, DDX47, DDX52, and DDX53, as well as the helicase
domains of DDX25 and DDX41. While the physiological roles of
these proteins are diverse (Table 1) all structures show the RecA-like
fold.Superpositionofthe DEAD-domainstructuresgivesroot mean
square deviations of Ca-atom positions between 0.6 and 1.9 A˚for
proteins with sequence identity between 86 and 27%. The two
helicase domains have a sequence identity of 23% and their
structures superimpose with an r.m.s.d. of 3 A˚. Details of the
synchrotron data collection, structure determination, and refine-
ment statistics are presented in Table 2.
Superpostition of the different crystal structures illustrates the
location of flexible regions (Figure 1A, 1B). In general, regions of
high sequence conservation (the conserved motifs in particular)
contribute to the binding sites for nucleotide and for RNA, and
these sites coincide with the highest structural similarity (Figure 2).
Conversely, unconserved regions in the DEAD-domains deter-
mined here show a higher r.m.s.d. in their Ca-atom positions.
Some of the unconserved regions in the structures are flexible, as
documented by high B-factors and partially missing electron
density.
Diverse surface properties among DEAD domains
We compared the surface charge distributions of the DEAD-
domain structures (Figure 1E, 1H). All DEAD-domains feature a
conserved patch that constitutes the nucleotide binding site and
part of the RNA binding site. This patch forms a negatively
charged channel between a-helices 8 and 210 that extends to the
Mg2+-binding site. The negative charges originate from the side
chains of the two helices, including the DEAD-motif on a-helix 8.
As expected, the RNA binding cleft is positively charged in all
DEAD domains, but the charged patches differ in size. The
remainder of the DEAD-domain surfaces differs in electrostatic
surface properties among the family members.
ATP binding site: The flexible P-loop
Conserved motifs I (the P-loop), Ia, II, and the Q-motif
participate in nucleotide binding [3,23]. The P-loop and motif II
coordinate the nucleotide phosphates and the magnesium ion,
whereas residues of the Q-motif bind and recognize the adenine
moiety. The side chains that participate in nucleotide and
magnesium binding are highly conserved (Figure 2). The
nucleotide phosphates interact with backbone atoms, a conserved
lysine, and the divalent cation. Superposition of the DEAD-
domains shows that the structures of the P-loop and motif III are
determined by the state of nucleotide hydrolysis. The P-loop is in a
wide-open conformation when ATP is bound, as seen in DDX20
as well as in the previously published structures of DDX19 [22]
and eIF4AIII [19]. In the crystal complexes with either ADP or
AMP the loop closes up, resulting in a shift in Ca-atom positions
by up to 3.5 A˚between the ATP- and the AMP-states, or by up to
2.5 A˚between the ADP- and the AMP-state (Figure 3A). Thus the
conformation of the P-loop is determined by the nucleotide
phosphates, and longer phosphate tails result in a more open loop.
Table 1. Summary of previously established roles and functions for the RNA helicases covered in this study.
HelicaseFunction
DDX2ADDX2A (eIF4A1) is essential for translation initiation. It is part of the eIF4F complex that consists of eIF4G, eIF4E and eIF4A [51–53]. Its activity is
strongly enhanced by eIF4G, eIF4B and eIF4H [54]. The eIF4F complex and eIF4A are potential targets for anti cancer drugs [55–57].
DDX2BAlso known as eIF4A2, an isoform of DDX2A.
DDX5 DDX5 is a co-regulator of different transcription factors including ERa, p53, MyoD and Runx2, but ATPase/helicase activity is not required for
transcriptional co-regulation. DDX5 also participates in pre-RNA processing, alternative splicing, microRNA and ribosomal RNA processing
(reviewed in ref. [6]).
DDX10DDX10 is probably involved in ribosome assembly. Fusion of the nucleoporin gene NUP98 with the DDX10 gene leads to the NUP98-DDX10 gene
product. This fusion protein is involved in leukemogenesis [58,59].
DDX18DDX18 (Myc-regulated DEAD-box protein, or MrDP; [60]) is a nucleolar protein that is specifically upregulated in highly proliferating cells [61].
DDX20DDX20 (Gemin3) is a component of the SMN (Survival of Motor Neurons) complex that is involved in assembly and reconstruction of different RNP
(ribonucleoprotein) complexes [62]. DDX20, Gemin4 and eIF2C2 form a separate complex that contains numerous miRNAs [63]. DDX20 also binds
to the Epstein-Barr Virus Nuclear Proteins EBNA2 and EBNA3C. The poliovirus-encoded proteinase 2Aprocleaves DDX20 resulting in DDX20
inactivation and reduced snRNP assembly [64].
DDX25DDX25 (GRTH) is a testis specific, gonadotropin and androgen regulated protein that is essential for completion of spermatogenesis [65]. DDX20
acts as a shuttling protein in the gene-specific nuclear export of RNA messages. Furthermore it regulates the translation of specific genes in germ
cells [66].
DDX41 DDX41 (Abstrakt) post-transcriptionally regulates the expression levels of the insc protein that is essential for control of cell polarity and spindle
orientation [67].
DDX47DDX47 is involved in pre-rRNA processing. It interacts with NOP132 which recruits pre-rRNA processing proteins to the region within the nucleolus
were rRNA is transcribed [68].
DDX52DDX52 (Rok1) is required for the release of snR30 (small nucleolar RNA-30) from pre-ribosomes. snR30 is one of three snoRNAs that are critical for
pre-rRNA processing in yeast. DDX52 ATPase activity is important for optimal pre-ribosomal RNA processing, but not essential for release of snR30
[69].
DDX53DDX53 (CAGE) is expressed in testis and various tumors, but not in other tissues. Expression of the CAGE-gene is determined by its methylation
status [70].
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Table 2. Summary of crystallographic data analysis and refinement statistics*.
Structure
DDX2A
DDX2B
DDX5
DDX10
DDX18
DDX20
DDX25
DDX41
DDX47
DDX52
DDX53
Domain
DEAD
DEAD
DEAD
DEAD
DEAD
DEAD
helicase
helicase
DEAD
DEAD
DEAD
PDB entry
2G9N
3BOR
3FE2
2PL3
3LY5
3B7G
2RB4
2P6N
3BER
3DKP
3IUY
Ligand
-
-
ADP
ADP
PO4
AMPPNP
-
-
AMP
ADP
AMP
Beamline
ESRF ID14-2
APS 19-ID
BESSY 14.2
MAX II I911-2
DIAMOND I04
ESRF ID29
BESSY 14.2
ESRF ID14-4
ESRF ID29
ESRF ID23-1
ESRF ID14-2
Wavelength (A˚)
0.93300
0.97242
0.9184
1.04123
0.9789
1.00595
0.95373
1.04005
0.97472
1.00000
0.97930
Space group
P 1 21 1
P 21 21 2
C 2 2 21
P 61 2 2
P 31
P 31 2 1
P 43 21 2
P 65 2 2
C 1 2 1
P 1 21 1
P 1 21 1
Cell dimensions
a, b, c (A˚)
a=47.8,
b=78.25,
c=59.09
a=58.09,
b=80.1,
c=42.74
a=84.57,
b=106.87,
c=117.32
a=63.5,
b=63.5,
c=304.01
a=41.34,
b=41.34,
c=230.54
a=63.56,
b=63.56,
c=214.6
a=70.31,
b=70.31,
c=187.12
a=68.01,
b=68.01,
c=305.6
a=93.05,
b=70.37,
c=35.86
a=40.63,
b=38.36,
c=73.84
a=56.44,
b=61.25,
c=65.79
a, b, c (u)
a=90, b=103.43,
c=90
a=90, b=90,
c=90
a=90, b=90,
c=90
a=90, b=90,
c=120
a=90, b=90,
c=120
a=90, b=90,
c=120
a=90, b=90,
c=90
a=90, b=90,
c=120
a=90, b=90.7,
c=90
a=90, b=90.37,
c=90
a=90, b=96.36,
c=90
Highest resolution
shell range(A˚)
2.37–2.25
1.92–1.85
2.67–2.60
2.30–2.15
2.85–2.7
2.00–1.90
2.90–2.80
2.80–2.60
1.50–1.40
2.20–2.10
2.53–2.40
Rmeas(a)
0.07 (0.24)(b)
0.14 (0.67)(c)
0.14 (0.74)
0.05 (0.16)
0.24 (1.29)
0.081 (0.13)
0.11 (0.82)(b)
0.10 (0.45)
0.05 (0.14)
0.09 (0.38)
0.20 (0.61)
I/s(I)
7.4 (5.3)
27.6 (3.8)(d)
11.0 (2.4)
54.1 (24.5)
6.2 (2.0)
32.10 (21.3)
15.65 (2.6)
35.9 (12.6)
28.7 (14.1)
12.8 (4.6)
13.4 (4.2)
Completeness (%)
99.7 (100.0)
100 (99.9)
99.4 (99.8)
99.7 (100.0)
99.98 (100.0)
99.7 (100.0)
100.0 (100.0)
99.9 (100.0)
99.5 (100.0)
99.5 (99.8)
100.0 (100.0)
Redundancy
7.4 (3.5)
11.4 (10.1)
4.0 (4.0)
25.4 (26.5)
5.1 (5.5)
20.5 (18.2)
8.8 (9.1)
40.0 (41.8)
7.3 (7.3)
3.7 (3.7)
9.3 (9.5)
RefinementResolution range (A˚)
20–2.25
30–1.85
35–2.6
30–2.15
38.4–2.8
38.43–1.90
19.44–2.80
29.72–2.60
28.24–1.40
19.56–2.10
45.13–2.40
No. reflections
19100
17560
15770
19842
10358
38606
11208
13220
43193
12804
17613
Rwork/Rfree
0.1761/0.2573
0.185/0.226
0.205/0.273
0.210/0.248
0.246/0.274
0.172/0.202
0.233/0.267
0.243/0.294
0.165/0.189
0.182/0.236
0.201/0.251
B-factor (A˚2)
Protein
15
21
27
48
47
18
82
57
13
23
23
Water
17
17
20
45
29
29
53
28
17
25
Ligand
27
43
36
26
14
12
17
R.m.s deviations
Bond lengths (A˚)
0.018
0.015
0.012
0.017
0.007
0.014
0.01
0.016
0.014
0.013
0.007
Bond angles (u)
1.732
1.344
1.354
1.695
0.952
1.648
1.244
1.602
1.638
1.414
1.157
Ramachandran plot
Favored regions (%)(e)
96.0
99.5
98.3
98.2
96.88
98.8
91.4
95.2
99.6
99.6
98.8
Allowed regions (%)(e)
99.2
100
100
100
100
100
99.7
100
100
100
100
*Values in parentheses refer to the outermost resolution shell.
(a)Rmeasas described in [71].
(b)Rsymas described in [72].
(c)Calculated using Rmerge, Version 2 [73].
(d)Calculated as the ratio of average I over average error.
(e)Determined using Molprobity [74].
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This observation agrees with previous results [24]. Motif III
follows the P-loop transition and its position changes by up to 3 A˚
toward the P-loop. Motifs Ia, Ib and II seem unaffected by the
state of ATP hydrolysis, and their conformations remain
unchanged even in the crystal structures in which the nucleotide
binding site is not occupied.
Two of the structures show unique P-loop conformations. The
DDX2B structure features an a-helix 4 that is longer than in other
helicases, and leads into an unusually closed P-loop conformation
(Figure 3C). As a consequence, the ATP binding site is not visible
on the surface of the DDX2B structure. This conformation is most
likely induced by a crystal contact in this region.
The AMPPNP-bound DDX20 structure contains no metal ion
(Figure 3B). Lack of c-phosphate coordination by a metal ion leads
to a shift in the position of the b- and c-phosphates, which bind
where the a- and b-phosphates are bound in other ATP
complexes. Since the adenine base is coordinated in the usual
fashion the a-phosphate and the sugar moiety are tilted out of the
expected positions. This illustrates that DDX20 (and presumably
other helicases) can bind ATP also in the absence of divalent
cation. However, a divalent cation is needed to allow coordination
of three phosphates in the correct geometry for catalysis.
Diversity in ATP coordination
Some of the side chains that interact with the nucleotides are
not conserved, and most of these are found in the Q-motif. Three
hydrogen bonds between the adenine ring and the protein ensure
specific binding of adenosine nucleotides. These are formed by the
conserved glutamine and the backbone carbonyl five residues
upstream of the glutamine (Figure 3). The 6thresidue upstream of
the conserved glutamine is an aromatic residue in most DEAD-
box helicases. Its side chain stacks with the nucleotide base,
stabilizing it in its position. Interestingly this residue is not
conserved: While phenylalanine is most common, DDX10 has a
tyrosine and DDX47 has a tryptophan in the corresponding
position. Moreover, an aromatic residue in this position is not
obligatory: DDX53 features an isoleucine, with weaker van-der-
Waals interactions with the adenosine ring than the base stacking
interactions with the aromatic side chains (Figure 3D). We
analyzed the protein-nucleotide binding interfaces in these crystal
structures using the PISA server [25]. This analysis showed that,
while the overall ligand interface areas are similar in the different
nucleotide complexes, the contribution by the base stacking
residues vary considerably. The variability in the stacking residue
position may reflect different needs for conformational flexibility in
this region of the DEAD-domains.
Helicase domain variation
The helicase domain contributes to nucleotide coordination via
motifs V and VI. From the closed state DDX19 structure [22] it is
apparent that four side chains are of particular importance: The
aspartate of motif V coordinates the O39 of the ribose. The second
arginine side chain of motif VI (HRxGRxGR) interacts with the c-
phosphate. The third arginine, which is also the putative arginine
finger during ATP hydrolysis, coordinates all three ATP
phosphates. The variable residue that follows this arginine
coordinates the adenosine ring by different means. In the
DDX19 helicase domain a phenylalanine stacks with the
adenosine rings. A superposition of DDX19 with the DDX25
and DDX41 helicase domains shows that in the latter two
structures part of motif VI is not visible in the electron density,
indicating its flexibility. The conserved motifs IV and VI
superpose well, whereas motif V shows different conformations
in all three structures (Figure 1B).
The only part of motif IV that is not flexible is the histidine-
arginine pair, and it superposes in all three crystal structures. The
arginine points to a negatively charged pocked formed in part by
side chains from motifs IV and V in the inside of the helicase
domain. The aliphatic part of the arginine side chain makes a
hydrophobic contact with the phenylalanine of motif IV. In the
two-domain closed state structures the histidine interacts with the
SAT motif from the helicase domain. Therefore, the SAT motif is
indirectly linked to the ATP binding site as well as to the RNA
binding sites of both domains. This explains the central
importance of this motif in the coupling of ATP hydrolysis and
RNA unwinding [26]. In SAT-motif mutants of eIF4A the
ATPase and helicase activities were uncoupled [27]: SAT-to-AAA
mutant protein is capable of binding RNA in an ATP dependent
manner, but lacks RNA unwinding activity.
Conserved and variable parts constitute the RNA binding
site
The available atomic resolution structures of DEAD-box
helicases with bound RNA [19–22] show that the DEAD-domain
contributes to RNA binding through two conserved and one
variable structural element: (i) Motif Ia; (ii) a-helix 7, with its
conserved motif Ib; and (iii) the variable loop connecting b-sheets 3
and 4. These interactions are illustrated for DDX19 in Figure 4A:
While the variable loop clamps the RNA substrate in a specific
conformation, motifs Ia and Ib each coordinate an RNA-backbone
phosphate and induce a tilt of one or more RNA bases.
Conserved motifs Ia and Ib of DDX19 and all DEAD-domain
structures described here superimpose perfectly (Figure 4). This
leads us to conclude that RNA substrates are bound in a similar
conformation by the conserved motifs of all these DEAD-domains.
The variability in part of the RNA binding sites (Figure 4D), on
the other hand, implies that different helicases could stabilize
specific RNA conformations. In addition, variable side chain
contribution may also reflect optimal recognition of specific
nucleotide sequences.
Inspection of the RNA complexes of DDX19, vasa, and
eIF4AIII [19–22] shows that the conserved motif that makes the
most extensive contacts with the RNA-backbone phosphates is
motif Ib. In two of our DEAD-domain crystal structures, anions
from the crystallization buffers are bound to motif Ib (a sulfate in
DDX5, and a phosphate in DDX47) highlighting the ability of this
motif to bind polyanions.
Figure 1. Crystal structures of DEAD-box conserved domains-1 and -2. (A) Superposition of the DEAD-domains of DDX2A (green), DDX2B
(brown), DDX5 (red), DDX10 (turquoise), DDX18 (grey), DDX47 (dark blue), DDX52 (yellow), and DDX53 (dark yellow). The positions of conserved
motifs I–III (black) are indicated. (B) Superposition of the helicase domains of DDX19 (light blue), DDX25 (grey) and DDX41 (orange). The positions of
conserved motifs IV–VI (black) are indicated. (C) Cartoon representations of the DDX5 helicase domain in the same orientations as in the following
two panels. (D) Conserved surface patches (green), projected onto the DDX47 DEAD-domain surface. (E) Electrostatic surface representation of DEAD-
domains. Negative charges are shown in red and positive charges in blue. (F) Cartoon representations of the DDX41 helicase domain in the same
orientation as in the following two panels. The RNA and AMPPNP (sticks representation) of the superposed DDX19 structure mark the RNA and
nucleotide binding sites. (G) Conserved surface patches (green), projected onto the DDX25 helicase-domain surface. (H) Electrostatic surface
representation of helicase domains.
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Figure 2. Sequence alignments of the two RecA-like domains of the DEAD-box proteins described in this study. Conserved sequence
motifs are indicated. Secondary structural elements are given for DDX19 (PDB entry 3G0H) above the alignment. Asterisks mark the terminal aspartate
of the DEAD motif and the arginine of motif V, the interaction of which is central to positioning of a-helix 8 (see also Figure 5C, D). Sequences shown
are human DDX19B (gene accession number: 13177688); DDX10 (13514831); DDX18 (38327634); DDX20 (23270929); DDX25 (29792166); DDX41
(21071032); DDX47 (45786091); DDX5 (16359122); DDX52 (27697141); DDX53 (45709415); eIF4A1/DDX2A (16307020); and eIF4A2/DDX2B (45645183).
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Mechanism for unblocking of the RNA binding site
Our crystal structures of both DEAD-domains and helicase
domains in isolation reveal that the RNA binding site on each
domain is in a conformation that is incompetent to bind RNA
substrate. In the free helicase domain structures motif V, an
important RNA backbone interaction site [19–22] is in a
binding incompetent conformation. In the closed state, an
RNA binding competent conformation is stabilized by the
interaction of the conserved arginine of motif V with the C-
terminal aspartic acid of the DEAD-motif (Figure 5C). In all
single DEAD-domain structures, a-helix 8 has adopted a
position that would block the RNA binding site. By contrast,
upon cleft closure in the two-domain ATP analog and RNA
complexes, a-helix 8 has moved out of the RNA binding site
(Figure 5).
Thus, superposition of single DEAD-domain structures onto
the closed state structures of DDX19 and eIF4AIII suggests
involvement of a-helix 8 in the formation of a competent RNA
binding site. How is a-helix 8 displaced to allow access to the
RNA substrate binding site? No direct interaction between a-
helix 8 and the RNA have been observed; thus displacement of
a-helix 8 by the RNA substrate itself seems unlikely. Also,
binding of ATP itself cannot cause a-helix 8 rotation out of the
RNA site: The DEAD-motif is the only link between the
nucleotide and a-helix 8, but the state of nucleotide hydrolysis
does not influence the conformation of the DEAD motif (motif
II; Figure 1A, 3A).
Instead, we propose direct involvement of the helicase domain
in the activation of the RNA binding site on the DEAD-domain:
In the complex structures, the conserved arginine of motif V in
the helicase domain forms a salt bridge with the C-terminal
aspartic acid of the DEAD-motif, which is also the terminal
residue of a-helix 8 (Figure 5C, 5D). This interaction stabilizes a
conformation where a-helix 8 is rotated out of the RNA binding
site (Figure 5D). We propose that ATP binding primes the
helicases for RNA substrate binding by bringing the domains
together to allow motif V to push a-helix 8 out of the RNA site
on the DEAD-domain. RNA binding to the DEAD-domain then
completes cleft closure to allow formation of an active ATPase
site (Figure 6).
This model of cleft closure and helicase activation through
regulation of a-helix 8 can reconcile published data. Moreover, it
can explain how substrate release in the post-hydrolysis state is
achieved. DEAD-box helicases typically bind ADP with higher
affinity that ATP [28–31], and binding of ATP and RNA are
cooperative [31–35]. Thus, the binding energy of the RNA-
protein interaction likely stabilizes a strained conformation that is
competent for ATP hydrolysis. Conversely, relief of this strain
upon ATP hydrolysis and phosphate release likely drives RNA
substrate remodeling [36]. According to our comparative
structural analysis, ATP hydrolysis and phosphate release would
allow a-helix 8 to move back into its original position, releasing the
RNA substrate and switching back to a binding incompetent RNA
site on the DEAD domain.
Figure 3. Details of the ATP binding sites. (A) Superposition of multiple DEAD-domains to illustrate variability in P-loop (Motif I) conformations.
P-loops in DEAD-domain structures with bound phosphate (yellow), with bound AMP (orange), with bound ADP (red), DDX19 P-loop with bound
AMPPNP and Mg2+(blue), DDX20 P-loop with bound AMPPNP (magenta), and P-loop in nucleotide-free eIF4A/DDX2A (green) are shown. Motifs I, II
and III are indicated. (B) Two different conformations of the b- and c-phosphates in the DDX20-AMPPNP complex. Side chains that interact with the
AMPPNP are shown as balls-and-sticks. (C) DDX2B with a closed P-loop. The a-helix that follows the P-loop starts one turn earlier compared to other
DEAD-domain structures shown. (D) Variability of interactions with the adenosine nucleotide. The adenosine moiety is coordinated through p-
stacking interactions or hydrophobic interactions. Numbers denote the interaction surface, in A˚2, between the nucleotide and the stacking side chain,
as determined using the PISA server [25].
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DExH-box RNA helicases differ in some aspects from the
DEAD-motif containing helicases. The hepatitis C virus DExH-
box helicase NS3 binds RNA in the absence of ATP [37]. DExH
helicase NPH-II unwinds RNA in a processive fashion [38] and
thus stays bound to the RNA after each unwinding step. Our
model for the role of a-helix 8 in cleft closure of DEAD-proteins
is consistent also with these properties of DExH-box RNA
helicases. Whereas a-helix 8 is conserved in all DEAD-box
proteins, it is missing in the DExH-box proteins (refs. [37,39–42]
and references therein). Moreover, the DEAD-motif aspartic acid
side chain that mediates opening of the RNA binding site
(Figure 5) is replaced by the histidine of the DExH-motif. Thus
apparently, in the absence of a-helix 8 that may block the RNA
site, this terminal aspartic acid is redundant, and the histidine
that substitutes it fulfills a different function [42]. We conclude
that DEAD- and DExH-box helicases differ significantly in the
coupling of the RNA binding event to the conformational cycle of
the two RecA domains.
Materials and Methods
All proteins were expressed in Escherichia coli as N-terminally
hexahistidine tagged fusion proteins, and purified by nickel affinity
chromatography and gel filtration. Proteins were crystallized in
sitting drops at 4uC or 20uC. X-ray diffraction data were collected
at the APS (Chicago, USA), the BESSY (Berlin, Germany), the
Diamond (Oxfordshire, UK), the ESRF (Grenoble, France), and
the MaxLab (Lund, Sweden) synchrotron radiation facilities. Data
were indexed and integrated using XDS [43], MOSFLM [44], or
DENZO [45], and scaled using XSCALE [43], SCALA [46] or
SCALEPACK [45]. Structures were solved by molecular replace-
ment using PHASER [47] or MOLREP [48], and refined using
Figure 4. RNA binding cleft on DEAD domains. (A) DDX19 (light blue; PDB entry 3G0H) with bound RNA (light orange). RNA-interacting side
chains are shown. (B) Flexible regions in DDX2B, DDX10 and DDX53 for which the electron density was not visible. (C) Sequence conservation in the
RNA binding cleft, mapped onto the DDX47 structure (red, conserved; orange, partly conserved). (D) RNA binding sites of selected DEAD-domains to
illustrate their sequence variation.
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Figure 5. Details of the RNA binding cleft. (A) DDX19 closed state structure (PDB entry 3G0H). DDX19-bound RNA, Mg2+-ion and AMPPNP are in
orange. (B) Superposition of several DEAD domain structures showing a conserved conformation of a-helix 8. (C) Interactions between the DEAD and
helicase domains of DDX19. (D) ‘‘Top-down’’ view of the open and closed RNA binding cleft. DDX5 (red), the ATP-state of DDX19 (blue) and DDX41
(orange) are shown. RNA (superposed from the DDX19 complex structure) is shown in light orange. (E) Surface representation of the DDX19-RNA
complex. Note that a-helix 8 does not come in contact with the RNA substrate. (F) Surface representation of DDX5 and the superposed RNA from the
DDX19 complex structure. Note that a-helix 8 would clash with the RNA substrate.
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REFMAC [49]. Refinement rounds were complemented with
manual rebuilding using COOT [50].
Detailed Materials and Methods can be found in Table S1.
Accession codes
The coordinates have been deposited in the Proteins Data Bank
with accession codes 2G9N, 3BOR, 3FE2, 2PL3, 3LY5, 3B7G,
2RB4, 2P6N, 3BER, 3DKP, and 3IUY.
Supporting Information
Table S1
and purification, crystallization, X-ray data processing.
Materials and methods detailing protein expression
Found at: doi:10.1371/journal.pone.0012791.s001 (0.15 MB
PDF)
Acknowledgments
We thank the beamline staff at the APS, BESSY, Diamond, ESRF, and
MaxLab synchrotron radiation facilities for excellent support. We would
also like to acknowledge our colleagues at the Structural Genomics
Consortium.
Author Contributions
Conceived and designed the experiments: LHS HWP MH MM HS.
Performed the experiments: PS TK SvdB RC LL MH LHS WT MH MM
AGT. Analyzed the data: PS TK LL MH HWP. Wrote the paper: PS HS.
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