Curmi1,2 1 School of Physics, University of New South Wales, Sydney, Australia 2 Centre for Immunology, St Vincent’s Hospital and University of New South Wales, Sydney, Australia 3 Depar
Trang 1redox-regulated chloride ion channel protein CLIC4
Dene R Littler1,2, Nagi N Assaad1,2, Stephen J Harrop1,2, Louise J Brown1,2,3, Greg J Pankhurst2, Paolo Luciani4, Marie-Isabel Aguilar5, Michele Mazzanti4, Mark A Berryman6, Samuel N Breit2 and Paul M G Curmi1,2
1 School of Physics, University of New South Wales, Sydney, Australia
2 Centre for Immunology, St Vincent’s Hospital and University of New South Wales, Sydney, Australia
3 Department of Chemistry and Biomolecular Sciences, Macquarie University, Sydney, Australia
4 Department of Cellular and Developmental Biology, University of Rome ‘La Sapienza’, Rome, Italy
5 Department of Biochemistry and Molecular Biology, Monash University, Clayton Victoria, Australia
6 Department of Biomedical Sciences, Molecular and Cellular Biology Program, Ohio University College of Osteopathic Medicine, Athens,
OH, USA
The chloride intracellular channels (CLICs) are a
recently discovered family of unusual intracellular
chloride ion channel proteins with six human family
members designated CLIC1 to CLIC6 The CLIC
pro-teins are highly conserved in vertebrates, with related
proteins in invertebrates Each CLIC protein contains
a conserved C-terminal 240-residue module which, in
the soluble form of the protein, is a structural
homo-logue of the glutathione S-transferases (GSTs) [1,2]
CLIC5B and CLIC6 both possess an additional large
hydrophilic N-terminal domain (170 and 440 residues,
respectively), not present in other CLICs
CLIC4 (also called mtCLIC, HuH1 and p64H1) was the first human CLIC to be identified [3] It is expressed in a wide variety of tissues and is highly con-served across species [4–8] The intracellular localiza-tion of CLIC4 varies considerably among different cultured cell lines and tissues, ranging from the plasma membrane [5,7,9] to various intracellular organelles including the inner mitochondrial membrane [10], the caveolae and trans-Golgi network [5], the ER [4] and large dense core vesicles [6] The cellular localization of CLIC4 appears to be intimately linked to membranes, scaffolding proteins and the cytoskeleton [7,9,11,12]
Keywords
CLIC4; glutathione S-transferase; ion
channel; redox regulation; X-ray
crystallography
Correspondence
P.M.G Curmi, School of Physics,
University of New South Wales, Sydney,
NSW 2052, Australia
Fax: +61 29385 6060
Tel: +61 29385 4552
E-mail: p.curmi@unsw.edu.au
(Received 29 June 2005, revised 29 July
2005, accepted 9 August 2005)
doi:10.1111/j.1742-4658.2005.04909.x
The structure of CLIC4, a member of the CLIC family of putative intracel-lular chloride ion channel proteins, has been determined at 1.8 A˚ resolution
by X-ray crystallography The protein is monomeric and it is structurally similar to CLIC1, belonging to the GST fold class Differences between the structures of CLIC1 and CLIC4 are localized to helix 2 in the glutaredoxin-like N-terminal domain, which has previously been shown to undergo a dra-matic structural change in CLIC1 upon oxidation The structural differences
in this region correlate with the sequence differences, where the CLIC1 sequence appears to be atypical of the family Purified, recombinant, wild-type CLIC4 is shown to bind to artificial lipid bilayers, induce a chloride efflux current when associated with artificial liposomes and produce an ion channel in artificial bilayers with a conductance of 30 pS Membrane bind-ing is enhanced by oxidation of CLIC4 while no channels were observed via tip-dip electrophysiology in the presence of a reducing agent Thus, recom-binant CLIC4 appears to be able to form a redox-regulated ion channel in the absence of any partner proteins
Abbreviations
CLIC, chloride intracellular ion channel; CLIC4(ext), fusion protein consisting of human CLIC4 where the last two residues are replaced by a
16 amino acid peptide; GST, glutathione S-transferase; NLS, nuclear localization sequence.
Trang 2Multiple stress inducers result in translocation of
CLIC4 to the nucleus, possibly via an internal nuclear
localization sequence 199KVVAKKYR206 [13]
The biological function of CLIC4 is currently being
elucidated Studies in Xenopus laevis show that CLIC4
is expressed early in embryogenesis and that it is
devel-opmentally regulated [14] In mammalian cell lines,
increases in CLIC4 gene expression have been linked
to differentiation of keratinocytes [8] and adipocytes
[15] as well as TGF-b1-mediated transdifferentiation of
fibroblasts into myofibroblasts [16] One of the most
interesting functions of CLIC4 is its involvement in
apoptosis [8,10,17,18]
Like other CLIC proteins, CLIC4 appears to have
both a soluble, GST-like form and an integral
mem-brane form, which is resistant to alkali treatment [4,5]
Proteinase K treatment of microsomes containing
CLIC4 results in a 27 kDa reduction in the size of the
protein, leaving a 6 kDa fragment [4] This supports
the hypothesis that the integral membrane form of
the protein has a single transmembrane region near the
N-terminus running from approximately Cys35 to
Val57 [4]
Experiments characterizing the channel properties of
CLIC4 have produced varying results Patch-clamp
studies of CLIC4-associated plasma membrane channel
activity in transfected human embryonic kidney
HEK-293 cells revealed an anion channel activity of around
1 pS conductance [19], while the incorporation of
vesi-cles containing purified CLIC4 from these cells into
lipid bilayers resulted in anion channel activity with a
conductance of 10–50 pS [4,19] The reason for the
dif-ference between these two results is not clear [20] The
inhibition of the CLIC4 conduction observed in
HEK-293 cells via antibodies indicates that the C-terminal
portion of the protein (residues 60–253) resides in the
cytoplasm [19] Similar experiments have demonstrated
that the integral membrane form of CLIC1 crosses the
membrane an odd number of times (most probably
once) leaving a cytoplasmic C-terminus and an exterior
N-terminus [21]
To date, structural studies on CLIC proteins have
focused on CLIC1 [1,22] The structure of the soluble
form of CLIC1 has been determined, showing that it
has a GST fold with a covalent binding site for
gluta-thione [1] More recently, CLIC1 has been shown to
undergo a major conformational change on oxidation
[22] In this reversible transition, the N-domain of
CLIC1 is completely rearranged, resulting in the
expo-sure of a large, hydrophobic surface, concomitant with
the formation of an intramolecular disulfide bond
between Cys24 and Cys59 In vitro, this new
confor-mation is stabilized by noncovalent dimerization We
have proposed that in vivo the conformation observed
in the oxidized state represents an intermediate mem-brane docking form [22]
Given that Cys59 is unique to CLIC1 (Ala70 in CLIC4), it is important to characterize the structure of CLIC4 and to determine whether its channel activity is redox-regulated In this paper, we report the 1.8 A˚ reso-lution crystal structure of the soluble form of a human CLIC4 with a C-terminal extension, CLIC4(ext), where the last two amino acids of CLIC4 have been serendipi-tously replaced by a 16-residue peptide The structure shows a two domain, GST-like protein, which is highly homologous to that of the soluble form of CLIC1 [1] Differences between the structures are analyzed using Ramachandran and real-space distance measures The observed differences are localized to the region around helix 2 (N-terminal domain), which, in CLIC1, under-goes a dramatic structural change induced by oxidation [22] Recombinant Escherichia coli expressed soluble, wild-type CLIC4 associates with lipid bilayers, as monitored by surface plasmon resonance, and at low
pH induces the efflux of chloride ions from artificial liposomes in a concentration-dependent manner The interaction between CLIC4 and the lipid bilayers is enhanced when CLIC4 is oxidized Tip dip electro-physiological recordings show that recombinant CLIC4 produces ion channels in artificial bilayers with a con-ductance of approximately 30 pS under nonreducing conditions while no channel activity was observed under reducing conditions (5 mm dithiothreitol) Thus, like CLIC1, recombinant CLIC4 appears to be capable of forming ion channels in synthetic bilayers under non-reducing conditions in the absence of any partner proteins
Results
Structure of the CLIC4
A fusion protein was accidentally constructed (due to
a PCR primer error), consisting of the human CLIC4 sequence where the last two residues, Thr252 and Lys253, were replaced by a 16-residue peptide (sequence: PSKVPKGEFQHTGGRY) The resultant protein, called CLIC4(ext), was highly soluble and monomeric in solution It crystallized in the space group P21212 and the structure of CLIC4(ext) was determined at 1.8 A˚ resolution, with one molecule per asymmetric unit (Fig 1; Table 1)
The structure of CLIC4(ext) (Fig 1A) closely resem-bles that of the soluble form of CLIC1 (Fig 1B) [1], and thus belongs to the GST superfamily CLIC4(ext)
is monomeric in the crystal and has approximate
Trang 3dimensions of 50· 40 · 20 A˚3 CLIC4 has two
domains: an N-terminal domain (residues 16–105) with
a thioredoxin fold, closely resembling glutaredoxin;
and an all a-helical C-terminal domain The observed
structure consists of residues 16–163 and 173–257 with
the break in density corresponding to the flexible foot loop between helix 5 and helix 6 (Fig 1A, bottom left), which is not ordered in the CLIC4(ext) structure This flexible loop is unique to the CLIC proteins and
it is not seen in the GSTs
The foot loop in both CLIC1 and CLIC4(ext) struc-tures appears to hinge at two residues that are con-served in all vertebrate CLIC sequences: Pro158 and Arg176 The side chain guanidinium group of Arg176 forms a charged hydrogen-bonding network with back-bone carbonyl oxygen groups from both sides of the foot loop (Fig 2A) An identical structure is observed for CLIC1 The foot loop does not appear to be pre-sent in the sequences of the invertebrate CLICs from Drosophila melanogaster (AAF48326), Anopheles gam-bia(EAA45365) and Schistosoma japonica (AAP06293), however, it may be present in the sequence of the Caenorhabditis elegansCLIC (AAQ75554)
In the CLIC4(ext) crystal structure, the N-terminal side of the foot loop is anchored via interactions with residues near the reactive Cys35 of a neighboring molecule Clear electron density was observed for Glu162, whose side chain forms hydrogen bonds with
C
D
Fig 1 Overall crystal structure of CLIC4 (A) Ribbon diagram showing the crystal struc-ture of CLIC4(ext), where the last two resi-dues of the wild-type CLIC4 sequence have been replaced by a 16 residue peptide (top left hand corner) (B) The structure of CLIC1
in the same orientation as CLIC4 in (A) (C)
A stereogram showing the C a trace of CLIC4 with every 10th residue numbered (D) A stereogram showing a superposition
of the backbone traces of CLIC4 (green) and CLIC1 (mauve).
Table 1 Data reduction and refinement statistics.
Completeness (1.9–1.8 A ˚ shell) 99.9% (99.9%)
Rmerge(1.9–1.8 A ˚ shell) 0.063 (0.58)
R factor (1.9–1.8 A ˚ shell) 0.195 (0.28)
Ramachandran plot b
a
From REFMAC V [35].bFrom PROCHECK [38].
Trang 4the backbone and side chain of Asn34 and the side chain of Lys24 This crystal contact stabilizes the structure of the leading side of the foot loop
The putative internal nuclear localization sequence (NLS) of CLIC4 (residues 199–206: KVVAKKYR) is located at the C-terminus of helix 6 (Fig 2B) Three basic residues, Lys199, Lys203 and Lys204 form the solvent exposed face of helix 6 near its C-terminus, while Arg206 is on the top of the molecule, in the loop leaving helix 6 and it points in the opposite direction
to the other basic residues (Fig 2B) The structure of the NLS is almost identical to that seen in CLIC1, with the exception that the residue equivalent to Lys199 is Gln188 in CLIC1
We note that in the crystal structures of NLS pep-tides binding to their target importin⁄ karyopherin family proteins [23–25], the NLS adopts an extended conformation so as to position the basic NLS residues into the appropriate binding pockets Thus, for folded CLIC4 to use this nuclear import machinery, the C-terminus of helix 6 is likely to have to partially unfold so as to allow interaction between its NLS and importin⁄ karyopherin protein
The C-terminal extension of CLIC4(ext) was import-ant for crystallization, as no crystals of the wild-type protein have been grown to date In the crystal struc-ture, an extended chain can be seen which includes res-idues Pro252 to Lys257 These resres-idues make a crystal contact with one face of a neighboring molecule which comprises b-strands 3 and 4 and helix 3 Ser253 makes backbone and side chain hydrogen bonds with the side chain of Glu97, while the backbone carbonyl of Ser256 makes a hydrogen bond to the side chain of Asn81 The intervening residues of the C-terminal extension interact with the neighboring monomer via hydrophobic contacts
Comparison with the structure of the soluble form of CLIC1
Human CLIC1 and CLIC4 share 67% sequence identity with a high degree of structural homology as demo-nstrated by a root mean square deviation (RMSD) of 0.77 A˚ between the Caatoms over residues 17–159 and 175–251 (Fig 1D) The backbone structures overlay well except for the region around helix 2 (including con-necting loops, Leu59 to His74) and the flexible foot loop (Leu159 to Thr175) For the most part, side chains adopt the equivalent rotamer in both structures For example, Trp218 on helix 7 is conserved in vertebrate CLICs with the exception of CLIC1, where it is replaced
by His208 (Fig 2C) and CLIC3, where it is replaced
by arginine In the structural overlay (Fig 2C), Trp218
A
B
C
D
Fig 2 Detailed views of the CLIC4 structure (A) Arg176 locks the
two ends of the foot loop into place via a network of hydrogen
bonds centered on it side chain guanidinium group (B) The NLS of
CLIC4 situated at the C-terminus of helix 6 and the subsequence
loop (C) Side chains in CLIC4 and CLIC1 adopt equivalent
rotam-ers Here Trp218 (CLIC4) and His208 (CLIC1) each stabilize the loop
connecting helices 6 and 7 by forming equivalent hydrogen bonds
to backbone carbonyl groups (D) An overlay of the loop connecting
helix 2 to b-strand 3 from CLIC4 (green and red) and CLIC1 (atomic
colors) All parts of this figure are in stereo.
Trang 5(CLIC4) and His208 (CLIC1) stabilize the loop
con-necting helices 6 and 7 by forming side chain hydrogen
bonds to the two carbonyl groups straddling Pro211,
which is conserved in all CLICs (including invertebrate
CLICs) except CLIC5 from X laevis (AAH56036)
where this residue is a serine
A more detailed comparison between the CLIC4
and CLIC1 structures is shown in Fig 3, which plots
the Ramachandran distances (blue) and real-space
dis-tances (red) for the Caatoms For the most part, there
are only minor differences in /–w angles between the
CLIC4 and the CLIC1 structures with major
differ-ences centered in three regions: the loop connecting
the C-terminus of helix 2 to b-strand 3; the hairpin connecting b-strands 3 and 4; and residues bounding the flexible foot loop
Just past the C-terminus of helix 2, there are two peaks in the Ramachandran distance plot (Fig 3) The first occurs in the loop between helix 2 and b-strand 3 representing the sequence 71-PGTHPP-76 (correspond-ing to the sequence 60-PGGQLP-65 in CLIC1) In both CLIC1 and CLIC4 the last proline of these sequences adopts the cis conformation, which is a con-served feature of thioredoxin fold proteins [26] This cis proline is adjacent to the redox site (Cys35 in CLIC4) and it has been shown to line the covalent
Fig 3 Structural comparison of CLIC4 with CLIC1 as a function of sequence The figure shows the Ramachandran distance (blue) and real-space distance (red) between equivalent residues in the CLIC4 and CLIC1 structures Below the graphs are the CLIC4 and CLIC1 sequences
in one letter code plus the secondary structure elements observed in both structures The highlighted residues show: yellow, conserved cys-teine residues; green, putative transmembrane domain; and blue, NLS.
Trang 6glutathione binding site in CLIC1 [1] In CLIC4 this
cisproline is preceded by a second proline, which is a
leucine in CLIC1 This sequence alteration appears to
result in a large rearrangement of the /–w angles for
the loop between Pro71 and Pro76 (Figs 2D and 3) A
change of 164 in the w angle of the proline at the
beginning of this loop region causes helix 2 to be
rota-ted by 15 with respect to helix 2 in the CLIC1
struc-ture This movement is evident in the real-space
distance plot (Fig 3) The double proline observed at
the C-terminus of this loop (N-terminal of b-strand 3)
is conserved in all vertebrate CLIC2, CLIC4, CLIC5
and CLIC6 sequences We note that in CLIC1 and
CLIC3 sequences the double proline is replaced by
Leu-Pro
The second Ramachandran distance peak occurs for
Asn81 and Ser82 occupying positions i + 1 and i + 2
within a type I¢ b-hairpin turn between b-strands 3
and 4 The corresponding b-hairpin turn in CLIC1
forms a type II¢ b-hairpin turn at residues Gly70 and
Thr71 CLIC1 is unique in having a Gly-Thr pair
within the b-hairpin while CLICs 2–6 all contain either
Asn or Asp followed by a Gly or Ser (Lys in CLIC2)
and are thus likely to adopt a type I¢ hairpin turn
similar to that of CLIC4
Both the Ramachandran and real-space differences
indicate that the observed parts of the foot loop differ
between the CLIC1 and CLIC4 structures (Fig 3) For
the CLIC1 structures [1,22], the foot loop differs
between each independent molecule where its
confor-mation appears to be dominated by crystal packing
interactions Thus, the foot loop is likely to be only
partially ordered in solution and differences between
the CLIC1 and CLIC4 structures within this region
are likely to reflect this flexibility
Membrane binding, liposome chloride efflux and
bilayer electrophysiology
Both the wild-type CLIC4 and CLIC4(ext) constructs
were tested for functionality with similar results The
proteins were assayed for lipid binding using surface
plasmon resonance measurements via a Biacore L1
chip that had been coated with unilamellar
phospha-tidylcholine liposomes Binding at neutral pH could
not be detected, however, a concentration dependent
binding was observed at lower pH values Figure 4A
shows the binding curves for 100, 200, 300 and
400 lgÆmL)1of wild-type CLIC4 at pH 5.0
Chloride efflux experiments have been used
previ-ously to test the functionality of recombinant CLIC1
[22,27] In the current experiments, CLIC4 was added
to a suspension of liposomes, which had been loaded
with 200 mm KCl CLIC4-dependent chloride efflux was triggered by the addition of the potassium iono-phore valinomycin In order to normalize the efflux results, the chloride efflux concentration is compared
to the total chloride concentration contained in the liposomes by rupturing the liposomes with detergent (Triton X-100) The percentage of chloride released is
pH dependent (Fig 4C), increasing at low pH This dependence resembles that observed for the channel activity of recombinant CLIC1 [28] The chloride efflux
is dependent on the concentration of CLIC4 (Fig 4D)
in a manner that is similar to that observed for CLIC1 [27]
CLIC4 was tested for channel activity via tip dip electrophysiology Recombinant CLIC4 was added to the bath solution of a tip-dip experimental apparatus
so as to reach a final concentration of 10 ngÆmL)1 After bilayer formation on the electrode tip, we waited until single channel activity was clearly detected where the experiment was carried on under a repetitive volt-age step of 50 mV and 500 ms duration We then used
a voltage steps protocol from )80 to +80 mV (20 mV steps) to obtain channel openings at each potential Amplitude histograms were used to calculate the exact single-channel size and the current values were used to build current⁄ voltage (i ⁄ V) relationships Linear regres-sion fit was used to interpolate experimental current amplitude at the different potentials Slope conduct-ance was calculated for different experiments In Fig 5, we show an example of current recordings (Fig 5A) and i⁄ V relationships (Fig 5B) for an experi-ment presenting current events with at least two levels Five independent experiments were analyzed and, in each case, we observed at least two conductance levels which we tentatively interpret as two independent channels The average conductance values for the three lowest current levels were 30.2 ± 1.4, 58 ± 2.1, and
86 ± 2.7 pS Given that these levels are approximately multiples of 30 pS, we tentatively interpret them as representing one, two and three independent CLIC4 channels, respectively
Redox regulation Given that CLIC1 channels are redox regulated, the effects of H2O2 oxidation and dithiothreitol reduction were tested on CLIC4 After incubation of CLIC4 with 2 mm H2O2 for 2 h at 18C, the protein con-tinued to run as a monomer on size exclusion chro-matography column (Superdex 75) This differs from the behavior of CLIC1, which forms a noncovalent dimer concomitant with the formation of an intra-molecular disulfide bond between Cys24 and Cys59
Trang 7This difference between CLIC1 and CLIC4 is not
unexpected since Cys59 in CLIC1 is not conserved in
other CLIC proteins and corresponds to Ala70 in
CLIC4
However, oxidation of CLIC4 via incubation with
0.4 mm H2O2 at room temperature for one hour
dra-matically increased its affinity for liposomes as
meas-ured by surface plasmon resonance (Fig 4B)
Furthermore, in the presence of 5 mm dithiothreitol,
no CLIC4 channel activity was observed in the tip dip
bilayer electrophysiology system (in 5 different
experi-ments, the current was recorded for one hour
alternat-ing holdalternat-ing potential between +50 and )50 mV every
minute) Thus, the channel formed by purified
recom-binant CLIC4 in artificial lipid bilayers appears to be
under redox control
Discussion
The structure of the soluble form of the CLIC4 mutant, CLIC4(ext) resembles that of CLIC1 as expec-ted from the high level of sequence identity (67%) Differences between the two structures are localized to helix 2 and surrounding loops in the N-domain and the flexible foot loop in the C-domain, with the latter being due to the flexibility of this region While the position of helix 2 and the preceding loop in CLI-C4(ext) differ from those observed in CLIC1, this can
be accounted for by rigid body displacement of this region However, the loop connecting helix 2 to b-strand 3 shows marked differences in /–w angles that appear to be related to sequence differences We note that the sequence of CLIC4 in this region is
Fig 4 Biophysical characterization of CLIC4 (A) Shows the surface plasmon resonance traces for the binding of CLIC4 (wt) to an L1 chip (Biacore) that has previously been coated with unilamellar liposomes so as to form a lipid bilayer The traces show the injection of BSA (1 mgÆmL)1) as a blocker, and subsequently, varying concentrations of CLIC4 (100, 200, 300 and 400 lgÆmL)1) coinjected with BSA (1 mgÆmL)1) The data shown are representative of two independent experiments (B) Shows surface plasmon resonance sensograms for the binding of both peroxide-treated and untreated CLIC4 to an L1 chip (BIAcore) that has previously been coated with unilamellar liposomes
so as to form a lipid bilayer The traces represent injections of 200 lgÆmL)1CLIC4 (either peroxide-treated or untreated) coinjected with BSA The data shown are representative of two independent experiments (C) pH effect on CLIC4 chloride efflux (30 lgÆmL)1CLIC4 final concentration) CLIC4 plus vesicles (n) or control buffer plus vesicles (m) were added to the required pH chloride-free buffer The percentage chloride release was measured 240 s after the addition of 1 l M valinomycin Triton X-100 was added (1% v ⁄ v) to normalize the chloride release from liposome vesicles (D) Effect of concentration on CLIC4 chloride efflux CLIC4 was added over the range of 3–27 lgÆmL)1final concentration Percentage chloride released was measured 240 s after the addition of 1 l M valinomycin.
Trang 8typical for all vertebrate CLIC2, CLIC4, CLIC5 and
CLIC6 sequences, thus, the structure observed in
CLIC4 is likely to be representative of the majority of
vertebrate CLICs
Like CLIC1, wild-type CLIC4 shows properties
that are consistent with the purified, soluble protein
being able to integrate into lipid bilayers and form an
ion channel in the absence of any accessory proteins
Our data show that CLIC4 binds to lipid bilayers,
induces the efflux of chloride ions from liposomes in
the presence of the ionophore valinomycin and
produ-ces channel events in a lipid bilayer as measured by
tip-dip electrophysiology The conductance of the base
channel (30 ± 2 pS) is similar to that observed for
purified CLIC1 under identical conditions (28 ± 9 pS
[22,28]) Together, these findings suggest that purified
recombinant, soluble CLIC4 can bind to lipid bilayers
and conduct a chloride ion current Thus, the
recom-binant wild-type CLIC4 protein appears to be
suffi-cient for ion channel activity at low pH in artificial
lipids
Our functional assays indicate that like CLIC1, the
CLIC4 ion channel activity is regulated by redox
con-ditions Oxidation of CLIC4 promotes binding to lipid
bilayers while no channel activity was observed in the
presence of the reducing agent, dithiothreitol, using
tip-dip electrophysiology Thus, it appears that under
the conditions tested so far, oxidation plays a key role
for the transition of CLIC4 from the soluble form to
an active integral membrane ion channel
Recently, we have shown that on oxidation CLIC1 adopts a conformation that differs significantly from the soluble, GST-like structure [1,22] This structural change in CLIC1 is stabilized by the formation of an intramolecular disulfide bond between Cys24 and Cys59, where the latter residue is unique to CLIC1 This conformation has been proposed to be the mem-brane docking form of CLIC1 This gives rise to two key questions First, does CLIC4 adopt a similar con-formation in order to dock with lipid bilayers? Second, does oxidation control channel activity in both CLIC1 and CLIC4 via a common mechanism?
To examine the first question, the residue Cys59 in CLIC1 corresponds to Ala70 in CLIC4 and is also an alanine in all other vertebrate CLIC sequences known
to date Thus, CLIC4 (as well as CLICs 2–6) cannot form a similar disulfide bond to stabilize the confor-mation observed in CLIC1 under oxidizing conditions However, this warrants further investigation because CLIC4 may still undergo a structural transition similar
to that proposed for CLIC1 during membrane dock-ing If CLIC4 did undergo such a transition, it may be either transient or else stabilized directly by interacting with the lipid bilayer
The differences between CLIC1 and the CLIC4(ext) structure in the loop preceding the conserved cis Pro76 may be relevant to the issue of structural transitions
In the structure of CLIC1, the region around Pro65 (equivalent to CLIC4 Pro76) acts like a hinge, facilita-ting the structural change observed on oxidation [22]
40
30
10
–20
–40
–60
Fig 5 Electrophysiological characterization
of CLIC4 Tip Dip experiment using wild-type
recombinant CLIC4 (A) Shows
single-chan-nel recordings at different membrane
poten-tials (reported on the right of each trace)
during a one second voltage step In this
experiment we observed at least two
cur-rent levels (B) Shows the curcur-rent⁄ voltage
relationship shows two distinct
conduct-ances From a linear regression fit we
calcu-lated the two different conductances of 31
(n) and 57 (d) pS.
Trang 9This region shows marked sequence and structural
dif-ferences in CLIC4 when compared to CLIC1 with the
CLIC4 sequence being typical of other CLIC proteins
(except for CLIC1 and CLIC3) It is possible that this
segment acts as a hinge in CLIC proteins other than
CLIC1, however, in these CLICs, any structural
change in this region would not be stabilized by the
formation of an intramolecular disulfide bond
To address the second question (common mechanism
for redox control of channel activity), both our current
and our previous experiments show that nonreducing
conditions are essential for recombinant CLIC1 or
CLIC4 to show channel activity [22] This implies that
in the absence of other proteins or cellular factors,
oxi-dation of the purified recombinant CLIC is necessary
for the formation of ion channels in synthetic lipid
bilayers If this oxidative activation mechanism is
shared by CLIC1 and CLIC4, then it must be linked to
one of the conserved cysteine residues (Cys35, Cys189
and Cys234 in CLIC4) rather than the formation of
the disulfide bond seen in the CLIC1 dimer [22]
The recently reported structure of the integral
mem-brane ClC chloride ion channel [29] cautions against
premature models of the CLIC channel Unlike the
other channels, the ClC structure does not show a
sim-ple pore structure consisting of a ‘hole through the
membrane’ Instead, the channel appears to consist of
two chloride binding sites inside the ClC dimer that
are accessible from either side of the membrane
Intriguingly, each ClC monomer is made up of two
structurally similar domains (presumably due to gene
duplication), each comprising approximately 250
resi-dues These domains interact in an antiparallel manner
to form the chloride channel
Our results show that CLIC4 is very similar to
CLIC1 in both its structural and its molecular
func-tion Like CLIC1, CLIC4 forms an ion channel whose
activity appears to be redox-regulated However, the
oxidation of CLIC4 does not stabilize the radical
con-formational change that we have observed in CLIC1
[22] Thus, key questions remain as to the precise role
of oxidation in controlling CLIC protein function
Experimental procedures
Cloning
To generate GST fusion proteins, cDNA encoding
full-length human CLIC4 (9) was amplified by PCR using
prim-ers that generated BamHI and either KpnI or HindIII
restriction sites at the ends The primers were sense:
CGCGGATCCATGGCGTTGTCGATGCCGC and
TGGC for wild-type CLIC4 The products were TA-cloned (Invitrogen, Inc., Carlsbad, CA, USA) and sequences veri-fied by DNA sequencing Plasmids were digested with BamHI and cloned into pGEX-2T (Amersham Pharmacia Biotech, Piscataway, NJ, USA)
Protein expression and purification
expressed and purified as described previously for CLIC1 with only minor modifications [1,22] E coli BL21 (DE3) cells containing the pGEX-2T plasmid and CLIC4(ext) or
recombinant CLIC4 was induced at an attenuance of
thio-b-d-galacto-side for 4 h The cells were then harvested and resuspended
in 30 mL phosphate buffered saline containing 10 mm
Bacterial lysate was prepared with two passes through a French pressure cell Triton X-100 was added to the lysate
with agitation The homogenate was then allowed to bind
to glutathione-sepharose 4B media (Amersham Biosciences)
as per the manufacturer’s instructions before washing with
300 mL phosphate-buffered saline (1 mm dithiothreitol) and equilibration in 20 mm Tris-base, 150 mm NaCl,
The bound fusion protein was then cleaved by thrombin at
a fusion protein–thrombin weight ratio of 50 : 1 for 16 h at room temperature
The eluted protein was then dialyzed into buffer A (20 mm Hepes, 50 mm NaCl, 1 mm dithiothreitol, 1 mm
DEAE-650(M) anion-exchange column pre-equilibrated in buffer
A The protein was eluted with a 300 mL gradient of buffer
A to buffer B (20 mm Hepes, 1 m NaCl, 1 mm
con-centrated and subsequently loaded onto a Superdex 75 column (Amersham Biosciences) pre-equilibrated with a buffer composed of 20 mm Hepes, 100 mm KCl, 1 mm
Crystallization
CLIC4(ext) crystals were obtained by the hanging-drop vapor-diffusion method Equal volumes (3 lL) of 14
placed over 1 mL of reservoir solution, which consisted of
Trang 100.2 m NH4F, 20% (w⁄ v) polyethylene glycol 3350 Crystals
grew at room temperature over a 2-week period
Data collection and processing
CLIC4(ext) crystals were progressively transferred into a
cryoprotectant solution consisting of reservoir solution and
flash-freezing and mounting at 100 K Diffraction data were
obtained at 100 K on a Mar345 image plate mounted on a
and Osmic confocal mirror optics The crystals diffracted
programs mosflm [30] and scala [31]
Structure determination and refinement
The CLIC1 monomer structure (1K0M) was used as a
molecular replacement probe using the CCP4 program
AMoRe [32] An initial phasing molecule consisting of
CLIC1 residues 6–165, 175–241 was used in the program
density map was clear and the CLIC4 sequence built
onto the original CLIC1 model in the program o [34]
This was refined using maximum likelihood methods
(program refmac v [35]) The final model consists of
residues 16–163 and 173–257 plus 158 water molecules
Residues Pro76 and Pro102 have cis peptide bonds The
reduc-tion and refinement statistics are summarized in Table 1
Residue Asp87 (N-terminus of helix 3) is in the
gener-ously allowed region of the Ramachandran plot, however,
its electron density is excellent The CLIC4(ext)
coordi-nates and structure factors have been deposited in the
Protein Data Bank (accession code 2AHE)
Ramachandran and real-space distances
To locate structural changes in an unbiased fashion, two
measures were used: the Ramachandran distance and a
real-space distance The Ramachandran distance, D, was
computed by comparing the Ramachandran plots for
CLIC1 and CLIC4(ext) using the equation:
The Ramachandran distance is measured in degrees
To compute the real-space distance, the CLIC4(ext) and
CLIC1 structures were superposed using the least squares
program lsqman [36] as implemented in the program o
[34] Using the superposed coordinates, the real-space
also computed
Membrane binding via surface plasmon resonance
Surface plasmon resonance experiments were carried out with a Biacore 2000 analytical system using the L1 sensor chip Methods were largely based on the protocol of Suba-singhe et al [37] Briefly, the chip surface was first cleaned with an injection of Chaps (40 lm) followed by an injection
of running buffer (10 mm phosphate, 10 mm Mes, 150 mm NaCl, pH 5.0) to ensure all detergent was removed from the system Small unilamellar liposomes of phosphatidyl-choline (soybean phosphatidylphosphatidyl-choline, Sigma P-5638), pre-pared by lipid extrusion, were then injected to generate a bilayer on the chip surface The surface was then briefly exposed to sodium hydroxide (10 mm) to remove any multi-lamellar structures Any remaining exposed surfaces of the
buffer) during the first phase of a coinjection BSA ± CLIC4 (100–400 lm) was then introduced in the second phase of the coinjection This strategy minimizes any possi-bility of nonspecific binding of CLIC4 After the coinjec-tion, the chip surface was stripped of all protein with an injection of 50 : 50 mixture of 100 mm HCl and isopropa-nol For oxidation experiments, dithiothreitol was removed from the protein sample using a PD-10 desalting column The protein concentration was measured by recording the
and incubated for 60 min at room temperature prior to coinjection with BSA as described above
Chloride efflux
Chloride efflux assay of CLIC4 channel activity was per-formed as described previously [22] Briefly, 400 nm
prepared by extrusion (Avestin Lipofast extruder) in 5 mm sodium phosphate buffer 200 mm KCl pH 6.0 Extravesicu-lar chloride was removed by desalting on Bio-Gel P6DG spin columns (Bio-Rad Laboratories Inc) equilibrated in
330 mm sucrose, 5 mm sodium phosphate at the required
pH (pH range 5.5–8.5) CLIC4 was also equilibrated into the same pH buffer and added to the liposomes in a total volume of 4 mL A chloride selective electrode (Radiometer Pacific) was used to monitor the potential driven chloride efflux from the vesicles upon the addition 1 lm valinomy-cin Triton X-100 was added to a final concentration of 1% after 240 s to normalize chloride release from vesicles
Electrophysiology
Single-channel recordings from lipid bilayers were obtained using the tip-dip method, as previously described [28] In brief, patch clamp pipettes (Garner Glass 7052) were made