Crystal structure of importin-abound to a peptide bearing
the nuclear localisation signal from chloride intracellular
channel protein 4
Andrew V. Mynott
1
, Stephen J. Harrop
1
, Louise J. Brown
2
, Samuel N. Breit
3
, Bostjan Kobe
4,5
and
Paul M. G. Curmi
1,3
1 School of Physics, University of New South Wales, Sydney, NSW, Australia
2 Department of Chemistry and Biomolecular Sciences, Macquarie University, Sydney, NSW, Australia
3 St Vincent’s Centre for Applied Medical Research, St Vincent’s Hospital and University of New South Wales, Sydney, NSW, Australia
4 School of Chemistry and Molecular Biosciences and Centre for Infectious Disease Research, University of Queensland, Brisbane, Qld,
Australia
5 Institute for Molecular Bioscience, University of Queensland, Brisbane, Qld, Australia
Introduction
The importin-a:bnuclear import pathway is one of the
best understood nuclear trafficking systems in the cell
[1]. The pathway operates via the importin-areceptor,
an armadillo (ARM) repeat protein, that recognizes and
binds directly to cargo protein in the cytoplasm. The im-
portin-a:importin-b:cargo complex travels through the
nuclear pore, with importin-bprimarily responsible for
negotiating passage through the nuclear pore complex.
This transport process is dependent on the ability of im-
portin-ato recognize specific nuclear localization signals
(NLSs) presented by the cargo protein. The acidic envi-
ronment of the importin-abinding sites confers a high
Keywords
chloride intracellular channel protein; CLIC4;
importin-a; nuclear localization signal (NLS);
nucleocytoplasmic transport
Correspondence
P. Curmi, School of Physics, University of
New South Wales, Sydney, NSW 2052,
Australia
Fax: +61 2 9385 6060
Tel: +61 2 9385 4552
E-mail: P.Curmi@unsw.edu.au
(Received 17 November 2010, revised 31
January 2011, accepted 23 February 2011)
doi:10.1111/j.1742-4658.2011.08086.x
It has been reported that a human chloride intracellular channel (CLIC)
protein, CLIC4, translocates to the nucleus in response to cellular stress,
facilitated by a putative CLIC4 nuclear localization signal (NLS). The
CLIC4 NLS adopts an a-helical structure in the native CLIC4 fold. It is
proposed that CLIC4 is transported to the nucleus via the classical nuclear
import pathway after binding the import receptor, importin-a. In this
study, we have determined the X-ray crystal structure of a truncated form
of importin-alacking the importin-bbinding domain, bound to a CLIC4
NLS peptide. The NLS peptide binds to the major binding site in an
extended conformation similar to that observed for the classical simian
virus 40 large T-antigen NLS. A Tyr residue within the CLIC4 NLS makes
surprisingly favourable interactions by forming side-chain hydrogen bonds
to the importin-abackbone. This structural evidence supports the hypothe-
sis that CLIC4 translocation to the nucleus is governed by the importin-a
nuclear import pathway, provided that CLIC4 can undergo a conforma-
tional rearrangement that exposes the NLS in an extended conformation.
Database
Structural data are available in the protein Data Bank under the accession number 3OQS.
Structured digital abstract
lCLIC4 and importin alpha bind by x-ray crystallography (View interaction)
Abbreviations
ARM, armadillo; CLIC, chloride intracellular channel; NLS, nuclear localization signal; RSCC, real space correlation coefficient; TAg, simian
virus 40 (SV40) large T-antigen.
1662 FEBS Journal 278 (2011) 1662–1675 ª2011 The Authors Journal compilation ª2011 FEBS
affinity to clusters of basic residues in the NLS. Mono-
partite NLSs consist of a single cluster of basic amino
acids, approximately six residues long, which generally
interact with the major binding site in importin-a. Struc-
tural studies have shown that an NLS binds importin-a
in an extended conformation, suggesting that functional
NLSs need to be unfolded and flexible within the cargo
protein. Recent studies have demonstrated an interaction
between importin-aand the chloride intracellular chan-
nel (CLIC) protein, CLIC4 [2,3].
The structure of a soluble form of CLIC4 shows
that it adopts the canonical glutathione S-transferase
fold with an N-terminal thioredoxin-like domain and
an a-helical C-terminal domain [4]. CLIC4 can form
poorly selective anion channels that are sensitive to
redox conditions [5] and, like other CLIC family mem-
bers, it is hypothesized that CLIC4 undergoes a struc-
tural transition from the soluble form to an integral
membrane form. CLIC4 is functionally important in
the cell and has recently been implicated in angiogene-
sis, with the observation that suppressed CLIC4
expression leads to the disruption of tubular morpho-
genesis [6]. CLIC4 is upregulated in human and mouse
differentiating cells [7] and has also been implicated in
the regulation of tumour growth [8,9].
In the event of cellular stress, CLIC4 translocates to
the nucleus in human osteosarcoma cells as well as
mouse S1 keratinocytes, where it is involved in an
apoptosis pathway independent of the apoptotic prote-
ase activating factor [2]. After translocation, CLIC4
localizes near the nuclear envelope and in the nucleo-
plasm. Immunoprecipitation experiments have shown
that tumour necrosis factor-aor etoposide treatment
of keratinocytes increases the constitutive interaction
between CLIC4 and various members of the nuclear
import machinery, including Ran, nuclear transport
factor-2 and importin-a[2]. Mutagenesis of a cluster
of basic residues in the putative CLIC4 NLS site
(199KVVAKKYR206 to 199TVVAITYG206) is suffi-
cient to prevent nuclear translocation, suggesting that
this monopartite NLS-like sequence has an active role
in the nuclear import process [2]. This indicates that
the binding of CLIC4 to importin-avia this putative
NLS is responsible for nuclear translocation; however,
in the crystal structure of soluble CLIC4, the putative
NLS adopts a helical conformation that would pre-
clude binding to importin-a(refer to Fig. 2D).
More recently, it has been shown that CLIC4
nuclear translocation is induced in mouse S1 keratino-
cytes by treatment with nitric oxide [3]. The nuclear
translocation is accompanied by S-nitrosylation of a
Cys residue in CLIC4, Cys234. The S-nitrosylation of
CLIC4 has been found to induce a conformational
change which destabilizes the native conformation.
Such a destabilization may facilitate the interaction
between the otherwise helical CLIC4 NLS and impor-
tin-a. It has been shown that S-nitrosylation of CLIC4
enhances the interaction with importin-a, as deter-
mined by immunoprecipitation [3].
In this article, we present the X-ray crystal structure
of mouse importin-a(70–529) bound to a peptide cor-
responding to the CLIC4 NLS. The importin-a(70–
529) construct used to obtain the importin-a:CLIC4
NLS complex lacks the first 69 residues that corre-
spond to the flexible importin-bbinding domain. The
importin-bbinding domain is known to have an
autoinhibitory function, whereby an internal NLS-like
sequence competes for the importin-abinding site,
reducing binding affinity for cargo proteins and help-
ing to facilitate the release of the cargo within the
nucleus [10,11]. The removal of the autoinhibitory
domain to create a truncated importin-aavoids possi-
ble competition for the binding site between this inter-
nal NLS and the CLIC4 NLS peptide.
The importin-aC-terminal domain (residues 70–529)
consists of 10 ARM structural repeats that form two
well-characterized cargo binding sites, referred to as
the major and minor binding sites [1]. These sites are
located in the concave face of the protein near regions
of invariant Trp and Asn arrays. The major binding
site spans ARM repeats 2, 3 and 4 (Fig. 1A). In the
major binding site, the positions of six NLS residues
are labelled P1–P6, following the directionality of a
bound NLS from the N-terminus to the C-terminus,
which runs antiparallel to importin-a.
Our structure shows that the CLIC4 NLS peptide
binds to the importin-amajor binding site in an
extended conformation consistent with a classical im-
portin-a:NLS complex. There is no clear interaction
between the CLIC4 NLS peptide and the minor bind-
ing site of importin-a. In the major binding site, elec-
tron density clearly defines the peptide residues
201VAKKYRN207, which have been included in the
final model with Lys203 occupying the critical P2 bind-
ing position. Our results reveal that Lys199 at the
putative CLIC4 NLS N-terminus is disordered in the
crystal and is therefore not necessary for peptide bind-
ing. The core binding pockets P2–P5 are occupied by
residues KKYR, a rather atypical NLS because of the
presence of a bulky aromatic Tyr residue in the P4
binding position. Surprisingly, the Tyr205 side-chain is
favourably placed at P4, forming hydrogen bonds with
the importin-amain chain. An analysis of normalized
Bfactors demonstrates a localized reduction in atomic
flexibility experienced by importin-aresidues as a
result of the binding of the CLIC4 NLS peptide.
A. V. Mynott et al.Crystal structure of importin-a:CLIC4 NLS peptide
FEBS Journal 278 (2011) 1662–1675 ª2011 The Authors Journal compilation ª2011 FEBS 1663
The importin-a:CLIC4 NLS structure presented in
this article adds to a growing body of knowledge on
the structural mechanisms that govern the classical
nuclear import model. It also clarifies that the CLIC4
NLS can indeed bind directly to importin-aon condi-
tion that it can unfold into an extended conformation.
Results
Structure of the importin-a:CLIC4 NLS peptide
complex
The structure of importin-a(70–529) bound to the
CLIC4 NLS peptide (198VKVVAKKYRN207) was
solved at 2.0 A
˚resolution using synchrotron radiation
(Table 1). The model of importin-ain the CLIC4 NLS
peptide complex includes residues 72–496 and closely
resembles the full-length importin-astructure that
incorporates the N-terminal autoinhibitory domain
(PDB:1IAL, rmsd of 0.20 A
˚across 425 C
a
atoms in
residues 72–496). The major binding site spanning
ARM repeats 2–4 has a similar conformation to the
equivalent region in apo importin-a(70–529), with an
rmsd of 0.16 A
˚(46 C
a
atoms) across the inner H3 heli-
ces, suggesting that there are minimal backbone con-
formational changes as a result of peptide binding.
Electron density corresponding to residues 201–207
(VAKKYRN) of the CLIC4 NLS peptide was unam-
biguously identified in the importin-amajor binding
site between ARM repeats 2–4. The F
o
)F
c
map con-
structed by omitting the peptide from model phases is
shown in Fig. 1A. The importin-aminor binding site
contains no electron density that unambiguously corre-
sponds to the CLIC4 NLS, and therefore no peptide
was modelled at this site. The CLIC4 NLS binds in an
extended conformation that runs antiparallel to impor-
tin-a, analogous with other NLS cargo. The average
atomic Bfactors for importin-ain the structure are
32.1 A
˚
2
for main-chain atoms, 35.7 A
˚
2
for side-chain
atoms and 33.8 A
˚
2
overall (3244 atoms). For the pep-
tide, Bfactors are slightly higher than those for
importin-a: 36.9 A
˚
2
for main-chain atoms, 40.2 A
˚
2
for
side-chain atoms and 38.7 A
˚
2
overall (62 total atoms).
Electron density analysis
Both the main-chain and side-chain atoms of the mod-
elled CLIC4 peptide show a good fit to the electron
density (Fig. 1B). The peptide residues 202–207 corre-
spond to the key binding positions P1–P6, with the
critical P2 position occupied by Lys203. This means
that the core basic motif, 203KKYR206, fills the
central binding pockets P2–P5 in which the majority
of peptide side-chain interactions take place with
importin-a. Therefore, the CLIC4 NLS satisfies the
accepted consensus sequence for an optimal NLS,
P2
K(K ⁄R)·(K ⁄R)
P5
[12]. The residue at P4, which has
been shown to contribute the least, energetically, to
peptide binding of the four main binding pockets [12],
is unambiguously occupied by Tyr205 as defined by
2F
o
)F
c
electron density.
The N-terminal peptide residues, 198VKV, are disor-
dered in the crystal and are thus likely to be highly
flexible. It is particularly noteworthy that the basic res-
idue, Lys199, defined as part of the putative CLIC4
NLS (KVVAKKYR) [2], does not contribute to pep-
tide binding. If the N-terminal flanking region
increases CLIC4 NLS affinity for importin-a,itis
unclear how it does so from our crystal structure. The
terminating carboxyl group of the peptide at Asn207 is
well defined in the 2F
o
)F
c
electron density map,
despite making no interactions with importin-a.
The presence of a Tyr residue at P4 is a strong indi-
cation that the bound peptide corresponds to the
CLIC4 NLS. There is also weak and unaccounted for
density approximately 4 A
˚from the aromatic plane of
Tyr205 in a position that may correspond to a cation–p
interaction. The cation in this case is likely to be an
Na
+
ion from the crystallization buffer, with a low
occupancy (< 50%). As a result of the weak nature of
the Tyr205 cation–pbond, it seems unlikely that it will
have a significant effect on the CLIC4 NLS peptide
binding to importin-a.
We have also analysed the veracity of the CLIC4
NLS model built in the major binding site by inspect-
ing the Fclic4nls
oFapo
odata–data difference Fourier (see
Materials and methods). This Fourier analysis reduces
bias when interpreting the density of a peptide bound
to importin-aand thus provides additional support for
our structure. The results are shown in Fig. 1D. As
expected, the electron density is strong along the pep-
tide main chain with well-defined carbonyl and amide
backbone groups. The one exception to this is the
location of the amide group of Lys204 at P3, where
there is a break in the main-chain density at the 2.8r
map level. The corresponding position in the apo struc-
ture has particularly strong density at this point, which
may correspond to a water molecule. Peptide side-
chain density is also well defined in the Fclic4nls
oFapo
o
map. At P1, the density is weak, but resembles Ala202.
The Lys residue at P2 (Lys203) is well defined despite
the presence of a partially occupied water at this
location in the apo structure. Lys204 at P3 and Arg206
at P5 are also well defined. Perhaps the most definitive
characteristic of the Fclic4nls
oFapo
odifference Fourier
is the strong and unambiguous electron density
Crystal structure of importin-a:CLIC4 NLS peptide A. V. Mynott et al.
1664 FEBS Journal 278 (2011) 1662–1675 ª2011 The Authors Journal compilation ª2011 FEBS
corresponding to Tyr205 at P4. There is a strong posi-
tive difference density peak near the Tyr205 O–C
f
bond at the 9.1rmap level, the strongest density peak
in the Fclic4nls
oFapo
odifference map. The presence of
Tyr205 is definitive evidence that the CLIC4 NLS
peptide binds importin-a(70–529).
CLIC4 NLS interactions with importin-a
The CLIC4 NLS forms an extensive network of inter-
actions with importin-athrough both main-chain and
side-chain atoms, similar to other importin-astructures
with a bound monopartite NLS [13–15]. Hydrogen
Fig. 1. The importin-a:CLIC4 NLS peptide complex. (A) The F
o
)F
c
‘omit’ electron density map over all atoms in importin-a. Positive con-
tours are shown at 2.8rin grey. Density corresponding to the bound CLIC4 NLS peptide is clearly visible in the major binding site. (B) Ste-
reoimage of the CLIC4 NLS peptide and 2F
o
)F
c
map. The CLIC4 NLS peptide bound to the importin-amajor binding site is shown as a
stick representation. Colour code for atoms: carbon, cyan; nitrogen, blue; oxygen, red. Electron density is contoured at 1.5rin grey. Binding
positions P1–P6 and the N- and C-termini are labelled. (C) Schematic representation of hydrogen bonds (broken lines, < 3.5 A
˚) between
importin-aand the CLIC4 NLS peptide,
P1
AKKYRN
P6
. Backbone carbonyl oxygens and amide nitrogens are shown as red and blue spheres,
respectively. Nitrogen and oxygen side-chain atoms are shown as blue and red squares, respectively. (D) Stereoimage of the CLIC4 NLS
bound to importin-a, showing the Fclic4nls
oFapo
odata–data difference Fourier map. Grey contours represent positive difference density at
2.8r. (E) Stereoimage of hydrogen-bond interactions (broken lines, < 3.5 A
˚). The CLIC4 NLS peptide is shown as a ball and stick represen-
tation, where carbons are black, nitrogens are blue and oxygens are red. Importin-ais shown in cartoon representation (cyan) with bonded
residues shown as sticks.
A. V. Mynott et al.Crystal structure of importin-a:CLIC4 NLS peptide
FEBS Journal 278 (2011) 1662–1675 ª2011 The Authors Journal compilation ª2011 FEBS 1665
bonding by the main chain of the CLIC4 NLS peptide
involves importin-aside chains in the conserved
WxxxN motifs of ARM repeats 2-4, which include res-
idues Trp142, Trp184, Asn146, Asn188 and Asn235
(Fig. 1C). In the CLIC4 NLS peptide, this corresponds
to hydrogen-bonded carbonyl and amide groups from
every second residue in the major binding site: P1
(Ala202), P3 (Lys204) and P5 (Arg206). The peptide
side chains in binding positions P2 (Lys203), P4
(Tyr205) and P5 (Arg206) form hydrogen bonds to the
importin-amain chain and side chains. In addition,
Lys203 forms a critical salt bridge with
impa
Asp192
(‘impa’ denotes importin-a; bond length, 2.80 A
˚;
N
f
)O
d
) at P2, the most energetically significant inter-
action involved in importin-arecognition of NLSs
[16,17].
Other basic residues in the peptide, Lys204 and
Arg206, fill negatively charged pockets at P3 and P5
Fig. 2. Analysis of the importin-a:CLIC4 NLS peptide complex. (A–C) Importin-ais coloured by the normalized Bfactor score, Bapo
z, over a
blue–magenta colour spectrum ()3rto +3r). (A) The bound CLIC4 NLS is shown on the molecular surface of importin-ain the major binding
site. (B) The importin-aC
a
backbone is shown as a cartoon tube representation in the same orientation as in (A). Important residues are
shown as a stick representation. (C) Full-length images of importin-acoloured by the Bapo
zscore. Residues in grey have not been included
in the calculation of Bapo
z. (D) The CLIC4 crystal structure (PDB:2AHE) is shown as a cartoon representation. The N-terminal thioredoxin
domain (blue) and C-terminal a-helical domain (green) are coloured separately. The CLIC4 NLS residues are highlighted in cyan. Inset: The
NLS is shown as a stick representation (carbons, cyan; oxygens, red; nitrogens, blue). Hydrogen bonds are represented by broken lines. (E)
A multiple sequence alignment of the CLIC4 NLS motif in human CLICs. Conserved residues are red, nonconserved residues are black and
perfect conservation is highlighted with red fill. The sequence of CLIC3 is added for comparison. Binding positions P1–P6 are shown in
an alignment corresponding to our importin-a:CLIC4 NLS peptide complex. Sequence alignment was performed using CLUSTALW [44] and
ESpript [45].
Crystal structure of importin-a:CLIC4 NLS peptide A. V. Mynott et al.
1666 FEBS Journal 278 (2011) 1662–1675 ª2011 The Authors Journal compilation ª2011 FEBS
