Biochem. J. (2010) 430, 315–324 (Printed in Great Britain)
315
doi:10.1042/BJ20100111
Interaction of iron regulatory protein-1 (IRP-1) with ATP/ADP maintains a
non-IRE-binding state
Zvezdana POPOVIC and Douglas M. TEMPLETON1
Laboratory Medicine and Pathobiology, University of Toronto, 1 King’s College Circle, Toronto, ON, Canada M5S 1A8
In its aconitase-inactive form, IRP-1 (iron regulatory protein1)/cytosolic aconitase binds to the IRE (iron-responsive element)
of several mRNAs to effect post-transcriptional regulation. We
have shown previously that IRP-1 has ATPase activity and
that binding of ATP suppresses the IRP-1/IRE interaction. In
the present study, we characterize the binding activity further.
Binding is observed with both [α-32 P]ATP and [α-32 P]ADP, but
not with [γ -32 P]ATP. Recombinant IRP-1 binds approximately
two molecules of ATP, and positive co-operativity is observed with
a Hill coefficient of 1.67 +
− 0.36 (EC50 = 44 μM) commencing
at 1 μM ATP. Similar characteristics are observed with both
apoprotein and the aconitase form. On binding, ATP is hydrolysed
to ADP, and similar binding parameters and co-operativity are
seen with ADP, suggesting that ATP hydrolysis is not rate
limiting in product formation. The non-hydrolysable analogue
AMP-PNP (adenosine 5 -[β,γ -imido]triphosphate) does not
induce co-operativity. Upon incubation of IRP-1 with increasing
concentrations of ATP or ADP, the protein migrates more slowly
on agarose gel electrophoresis, and there is a shift in the
CD spectrum. In this new state, adenosine nucleotide binding
is competed for by other nucleotides (CTP, GTP and AMPPNP), although ATP and ADP, but not the other nucleotides,
partially stabilize the protein against spontaneous loss of aconitase
activity when incubated at 37 ◦C. A mutant IRP-1(C437S) lacking
aconitase activity shows only one ATP-binding site and lacks
co-operativity. It has increased IRE-binding capacity and lower
ATPase activity (K m = 75 +
− 17 nmol/min per mg of protein)
compared with the wild-type protein (K m = 147 +
− 48 nmol/min
per mg of protein). Under normal cellular conditions, it is
predicted that ATP/ADP will maintain IRP-1 in a non-IREbinding state.
INTRODUCTION
[4Fe–4S] cluster [10,11]. In vitro, physiological concentrations
of ATP inhibit IRE/IRP-1 binding both in cell extracts and with
recombinant IRP-1. ADP has the same effect, in contrast with
the non-hydrolysable analogue AMP-PNP (adenosine 5 -[β,γ imido]triphosphate), indicating that in order to inhibit IRP-1binding activity, ATP must be hydrolysed [11].
In addition to regulation of IRP-1 by iron levels, its IREbinding activity is also affected by hypoxia, H2 O2 and oxidative
stress in general [12,13]. Each of these agents decreases
the level of intracellular ATP, while increasing IRP-1-binding
activity, suggesting a possible link between cellular iron levels
and energy metabolism [11]. Iron also modulates ATP levels, possibly through IRP-1-dependent translational control of mitochondrial aconitase [14]. Oexle et al. [15] demonstrated increased
mitochondrial oxygen consumption and ATP formation via
oxidative phosphorylation in iron-supplemented K562 cells,
and a subsequent reduction after iron deprivation. Furthermore,
IRP-1 may be regulated by phosphorylation/dephosphorylation
[16]. In the present study we further investigate the nature of
the interaction of ATP with recombinant IRP-1 in vitro. We
demonstrate co-operativity of adenosine nucleotide binding, and
present evidence for differential binding by different structural
states of the protein.
IRPs (iron-regulatory proteins) exert post-transcriptional regulatory control of expression of proteins involved in iron
homoeostasis. This control involves interaction of IRPs with
functional IREs (iron-responsive elements) in the 5 or 3
untranslated regions of mRNAs. Two IRPs have been identified:
IRP-1, which contains a [4Fe–4S] iron–sulfur cluster, and IRP-2,
which does not [1,2]. IRP-1 is generally believed to interconvert
between an enzymatically inactive IRE-binding state on the
one hand, and a non-binding form with a [4Fe–4S] cluster
and enzymatic aconitase activity on the other. A simple model
for the mechanism by which IRP-1 can sense iron involves
direct association of iron with an iron-depleted form of the
protein to form a complete [4Fe–4S] cluster. However, it is
well-established that the mitochondrion also plays an important
role in switching IRP-1 from IRE-binding activity to active
aconitase [3]. One reason is that the mitochondrion is the site of
cytosolic Fe–S cluster assembly. More than a dozen genes have
been implicated in Fe–S cluster biogenesis, and homologues of
some of them have been characterized in mammalian systems
[4,5]. Disruption of cluster assembly in mitochondria switches
aconitase activity of IRP-1 into IRE binding [6,7]. Evidence
from yeast and mammalian cells suggests that the cell does not
respond to total iron levels, but to the size of a regulatory iron
pool or perhaps to the flux of iron through the Fe–S assembly
pathway [3,8,9]. Furthermore, depleting ATP by uncoupling of
oxidative phosphorylation in mitochondria prevents the switch
of IRP-1 from the IRE-binding form to active enzyme with a
Key words: ATP hydrolysis, co-operative binding, iron-regulatory
protein, iron-responsive element, nucleotide binding.
EXPERIMENTAL
Purification of recombinant IRP-1
Plasmids pT7-his-IRP-1 [17] and pSG5-human IRP-1(C437S)
[18] (gifts from Dr Kostas Pantopoulos, Lady Davis Institute
Abbreviations used: AMP-PNP, adenosine 5 -[β,γ-imido]triphosphate; DTT, dithiothreitol; EMSA, electrophoretic mobility-shift assay; IRE, iron-responsive
element; IRP, iron regulatory protein; Ni-NTA, Ni2+ -nitrilotriacetate; rIRP-1, recombinant IRP-1.
1
To whom correspondence should be addressed (email [email protected]).
c The Authors Journal compilation c 2010 Biochemical Society
316
Z. Popovic and D. M. Templeton
for Medical Research, McGill University, Montréal, QC,
Canada) were cut with Pst1/BstEII restriction enzymes. A
1178 bp Pst1/BstEII fragment from mutant pSG5-human IRP-1,
encompassing the cysteine-to-serine mutation, was purified and
inserted into a 4 kb Pst1/BstEII fragment of pT7-his-IRP-1 to
give pT7-his-IRP-1(C437S). Both pT7 plasmids were grown in
Escherichia coli BL21 cells. Human recombinant IRP-1 (rIRP1) and rIRP-1(C437S) were purified from transformed E. coli
as described previously [17] and eluted from Ni-NTA (Ni2+ nitrilotriacetate) agarose beads with 50 mM imidazole. Purified
native and rIRP-1 appear to exist as an equilibrium of holoand apoprotein forms with intermediate (e.g. disulfide-bonded)
species also present [11,19,20]. In order to isolate holo- and
apoprotein for binding studies, several protocols of chemical
modification were evaluated. For the studies reported in the
present paper, active aconitase (holoprotein) was produced by
treating 50 μg of rIRP-1 in 100 mM Hepes (pH 7.4), 100 mM
DTT (dithiothreitol), with 1 mM ferrous ethylene ammonium
sulfate and 1 mM sodium sulfide for 1 h at room temperature under
argon [21]. The [4Fe–4S] cluster-free apoprotein was produced
by treating 50 μg of rIRP-1 with 100 mM DTT under alkaline
conditions (100 mM Tris, pH 8.8). Proteins were desalted on
protein desalting spin columns (Pierce) and equilibrated with
24 mM Hepes (pH 7.6), containing 150 mM potassium acetate,
1.5 mM MgCl2 and 5 % glycerol. IRE-binding activity was
tested with an EMSA (electrophoretic mobility-shift assay) as
described previously [11], using 0.5 μg of protein incubated with
30 ng of labelled IRE, and resolved on a 6 % non-denaturing
polyacrylamide gel.
Preparation of RNA transcripts
Transcription was performed in vitro with 1 μg of BamH1linearized plasmid pSPTfer [22], coding the human ferritin Hchain IRE as described previously [11], in the presence of 50 μCi
of [α-32 P]CTP (800 Ci/mmol; ICN) and T7 RNA polymerase,
using a Promega in vitro transcription system.
Filter-binding assay
ATP-binding activity was determined by a filter-binding assay
[23] with slight modifications. IRP-1 protein (1 μg) was incubated
with [α-32 P]ATP at room temperature (unless otherwise specified)
for 30 min in 20 μl of binding buffer [25 mM Tris (pH 7.6), with
100 mM KCl, 5 mM MgCl2 , 1 mM DTT and 10 % glycerol]
with different concentrations of ATP/Mg2+ . Competition experiments were performed in the presence of different concentrations
of unlabelled ATP, ADP, GTP, CTP or AMP-PNP. The nitrocellulose membrane was soaked briefly in 0.4 M KOH, rinsed
thoroughly, incubated in binding buffer and mounted on a dot-blot
apparatus. The binding reaction was stopped by addition of 75 μl
of ice-cold binding buffer and loaded on to the dot blot (30 μl in
triplicate wells). Each well was washed twice with 200 μl of icecold binding buffer. The membrane was dried, exposed to film,
and each spot cut out and counted in a liquid scintillation counter.
washed twice with 24 mM Hepes (pH 7.6), 150 mM potassium
acetate and 5 % glycerol. IRP-1 (5 μg) was incubated with
100 nM [α-32 P]ATP at room temperature for 30 min in 80 μl
of binding buffer [25 mM Tris (pH 7.6), with 100 mM KCl,
1 mM DTT and 10 % glycerol], with different concentrations
of unlabelled ATP/Mg2+ . The reaction mixture was mixed with
ATP-saturated aliquots of Ni-NTA and rotated for 30 min at room
temperature. Each tube was washed twice with 400 μl of 24 mM
Hepes (pH 7.6), containing 150 mM potassium acetate and 5 %
glycerol, and eluted with 400 μl of 24 mM Hepes (pH 7.6), with
150 mM potassium acetate, 5 % glycerol and 100 mM imidazole.
A blank tube contained no protein. Eluates were counted by liquid
scintillation.
IRE filter-binding assay
IRP-1 or IRP-1(C437S) (0.5 μg) was incubated with the indicated
amount of radiolabelled probe in EB buffer [10 mM Hepes
(pH 7.6), with 3 mM MgCl2 , 40 mM KCl and 1 mM DTT] for
30 min, and applied to a nitrocellulose membrane following the
ATP-binding protocol described above.
Preparation of [α-32 P]ADP
[α-32 P]ADP was prepared by hexokinase treatment of [α-32 P]ATP
(3000 Ci/mmol) (PerkinElmer). In total, 3 μl (10 pmol) of [α32
P]ATP was incubated at room temperature with 4 m-units/4 μl
hexokinase (1 m-unit/μl; Sigma), with 0.11 M glucose, 10 mM
MgCl2 and 30 mM Tris buffer (pH 8), for 60 min. Purity of [α32
P]ADP was confirmed by TLC. Aliquots (2 μl) were spotted
on to polyethyleneimine cellulose TLC plates (Sigma), resolved
using 0.5 M lithium chloride in 0.5 M formic acid, and visualized
by autoradiography. Conversion into ADP was quantitative as
determined by TLC.
Western blot analysis
For PAGE, treated IRP-1 is mixed with 50 mM Tris/HCl (pH 6.8),
with 100 mM DTT, 2 % SDS, 0.1 % Bromphenol Blue and
10 % glycerol, and boiled for 5 min. Proteins were resolved
on acrylamide gels (4–15 % gradient or 8 %) run in 25 mM
Tris, 192 mM glycine, with or without SDS. Separated proteins
were transferred on to nitrocellulose membranes, incubated with
an anti-IRP-1 antibody (Alpha Diagnostic International; 1:5000
dilution), and detected using ECL (enhanced chemiluminescence)
Western blot detection reagents (Amersham Bioscience).
Non-denaturing gel electrophoresis
Agarose gel (0.8 %) was made in 25 mM Tris/192 mM glycine
and electrophoresis was carried out in the same buffer.
Samples were loaded in 62.5 mM Tris/HCl (pH 6.8), with 40 %
glycerol and 0.01 % Bromophenol Blue, and run at 80 V. After
electrophoresis, gels were dried and exposed to X-ray film.
Ni-NTA agarose-binding assay
ATPase assay
In another set of experiments, nucleotide binding to recombinant
IRP-1 was followed by Ni-NTA agarose binding using a method
described by Obermann et al. [24]. Ni-NTA agarose beads were
pre-incubated (25 μl per sample) with 5 mM ATP/5 mM MgCl2
in 24 mM Hepes buffer (pH 7.6), with 150 mM potassium acetate,
5 % glycerol and 0.4 M KCl, for 30 min at room temperature and
ATPase assays were performed by measuring the release of
inorganic phosphate with Malachite Green amplification [25].
Protein (1.25 μg) was incubated in 100 μl of 25 mM Tris/HCl
(pH 7.6), containing 5 mM MgCl2 , 0.02 % Triton X-100, 1 mM
DTT and 0–2 mM ATP, for 30 min at 37 ◦C. Then 600 μl of
Malachite Green solution (0.17 % Malachite Green and 1.05 %
c The Authors Journal compilation c 2010 Biochemical Society
ATP binding to IRP-1
317
ammonium molybdate in 8.2 % HCl) was added for 1 min and
the reaction stopped with 75 μl of 34 % citric acid. Absorbance
was measured at 660 nm and compared with standard phosphate
solutions.
Aconitase activity
Aconitase activity was measured in a direct assay with 20 mM
cis-aconitate as the substrate. The disappearance of aconitate
was monitored spectrophotometrically at 240 nm, using a molar
absorption coefficient ε = 3600 M−1 · cm−1 [26,27]. Aconitase
activity is reported as the amount of substrate converted in
μmol/min per mg of protein at pH 7.4 and 25 ◦C.
CD
Recombinant protein in imidazole/Tris elution buffer was
transferred to 10 mM potassium phosphate (pH 8.1) by passage
through a PD10 Sepharose G-25 column (Amersham Biosciences)
and then concentrated in Microcon TM10 tubes (Millipore) to
approx. 3 μM. Triplicate spectra were recorded at 0.5 nm intervals
in a J-810 spectropolarimeter (Jasco) thermostatically controlled
at 20 ◦C, in a cuvette with a 0.2 cm path length.
Figure 1
Statistical analysis
Kinetic data and binding plots were fitted by non-linear regression
using GraphPad Prism (GraphPad Software). Significance of Hill
coefficients was determined using a one-sample Student’s t test
(GraphPad InStat).
Binding and hydrolysis of ATP by IRP-1
(A) Representative filter-binding assay of 1 μg of IRP-1 incubated with 50 nM [α-32 P]ATP or
[γ -32 P]ATP, both in the presence of 10 μM unlabelled ATP for 30 min at 25 ◦C. After incubation
for the times indicated, mixtures were blotted on to a nitrocellulose membrane and exposed
to X-ray film. (B) Time-dependence of ATP binding. Curves show the results of representative
filter-binding assays with 1 μg of IRP-1 incubated with 50 nM [α-32 P]ATP or [γ -32 P]ATP with
or without 10 μM ATP for up to 60 min at 25 ◦C. 䊉, 50 nM [α-32 P]ATP + 10 μM ATP; 䊊,
50 nM [γ -32 P]ATP + 10 μM ATP; 䊏, 100 nM [α-32 P]ATP only; 䊐, 100 nM [γ -32 P]ATP only.
After autoradiography, spots were cut from the nitrocellulose membrane and counted by liquid
scintillation. Curves were fit by non-linear regression.
RESULTS
ATP is hydrolysed after binding to rIRP-1
The presence of ATP at physiological concentrations (>1 mM)
inhibits IRP-1–IRE complex formation observed by EMSA, and
we demonstrated previously that IRP-1 binds ATP and has ATPase
activity [11]. To learn more about the IRP-1–ATP interaction, we
performed nucleotide-binding experiments with rIRP-1 using a
nitrocellulose filter-binding assay. At a concentration of 10 μM
ATP, binding of [α-32 P]ATP is readily detected, whereas binding
of label from [γ -32 P]ATP is negligible (Figure 1A). At 25 ◦C,
binding of [α-32 P]ATP reaches a plateau over 1 h, whereas [γ 32
P]ATP is still not observed (Figure 1B). This suggests that bound
ATP is hydrolysed rapidly upon binding.
ATP binding increased with IRP-1 concentration, saturating
at approx. 1 μM protein (results not shown). The binding is
effectively pH-independent across a physiological range, at
both 10 μM and 100 μM ATP (Supplementary Figure S1A at
http://www.BiochemJ.org/bj/430/bj4300315add.htm). It requires
the presence of Mg2+ , as substitution of MgCl2 in the binding
buffer by 10 mM EDTA eliminates binding, and addition of Mg2+
restores it (results not shown). Further studies were carried out at
pH 7.6 in stoichiometric solutions of ATP/Mg2+ .
To rule out filter binding as an artefact, e.g. due to precipitation
of IRP-1 and non-specific binding of ATP to the precipitate on the
filter membrane, binding was measured in an independent assay.
IRP-1 was incubated with [α-32 P]ATP and various concentrations
of unlabelled ATP up to 500 μM. IRP-1 was then bound
through its His tag to Ni-NTA agarose. Subsequent elution with
100 mM imidazole detached His-tagged IRP-1 with bound [α32
P]ATP (Supplementary Figure S1B). IRP-1 bound to Ni-NTA
agarose beads binds ATP with a Bmax of 3.74 +
− 0.78 and a K d
2
of 74.4 +
− 49.8 μM (R = 0.83), comparable with Bmax = 4.6 +
− 0.3
and K d = 86 +
− 17 μM reported previously [11]. Additional
evidence of specificity comes from the absence of binding when
BSA was substituted for IRP-1 in the filter-binding assay (results
not shown).
ATP-binding characteristics change with ATP concentration
We further characterized the ATP-binding interaction by increasing the concentration of unlabelled ATP while maintaining
the concentration of [α-32 P]ATP at 50 nM (Figures 2A and
2B). A 2–10-fold excess of unlabelled ATP partially competed
with binding of labelled ATP. However, at 1.25 μM, ATP
binding to IRP-1 changed dramatically (Figure 2A), increasing
approx. 50-fold. Analysis of IRP-1 binding to ATP in the 0.5–
200 μM range gave Bmax = 1.88 +
− 0.16 and K d = 54.8 +
− 12.5 μM
(R2 = 0.73). The corresponding Hill slope was 1.32 +
− 0.26
(R2 = 0.86) (Figures 2C and 2D), suggestive of co-operative
binding.
IRP-1 binds to ADP and ATP with similar characteristics
Previously [11] we demonstrated that ADP has the same inhibitory
effect on the IRP-1–IRE complex as ATP. Therefore we studied the
effect of ADP on [α-32 P]ATP binding in filter-binding assays (see
Supplementary Figures S2A–S2C at http://www.BiochemJ.org/
bj/430/bj4300315add.htm). Again, we detected competition of
radioligand binding at low concentrations of unlabelled ADP,
c The Authors Journal compilation c 2010 Biochemical Society
318
Figure 2
Z. Popovic and D. M. Templeton
Characterization of ATP binding
(A) Representative filter-binding assay of 50 nM [α-32 P]ATP in the presence of the indicated amounts of unlabelled ATP. (B) Non-linear regression analysis of binding data generated by cutting spots
from blots such as shown in (A). Values are means +
− S.D. from three separate experiments. Equilibrium binding parameters are reported in the text. (C) Hill plot of the data in (B). θ is the fraction of
ligand bound, calculated as [ATP] bound/n · [IRP-1], where the number of binding sites, n , is taken as B max calculated from (B). (D) Non-linear regression of data from (C) normalized to maximum
binding = 100 %, giving a Hill slope of 1.32 (R 2 = 0.86). See the text for details.
and at approx. 1 μM ADP binding again increased. Non-linear
regression analysis of [α-32 P]ATP binding in the presence of ADP
gave an apparent Bmax of 2.37 +
− 0.30 with a K d of 2121 +
− 30 μM
(R2 = 0.89). Again, a Hill slope of 1.39 +
0.35
(R
=
0.88)
was
−
consistent with co-operative binding.
Detailed examination of Hill plots
Representative experiments reported above give Hill slopes of
1.32 for ATP and 1.39 for ADP, corresponding to the illustrated
binding curves (Figure 2 and Supplementary Figure S2). These
indicate co-operative binding and are consistent with the sharp
increase in binding above 1 μM ATP. In fact, analysis of multiple
data sets over several months strengthened this conclusion. Seven
data sets for ATP binding were analysed and two failed to achieve
a plateau. Of the remaining five, S.D.s on the Hill coefficient
ranged from 6 to 14 %, and the mean value of 1.60 +
− 0.37 was
significantly greater than 1.0 (P = 0.02). With ADP, six data sets
were re-analysed. One showed no plateau and one showed a S.D.
of 54 %. Eliminating these two, the remaining four data sets gave
a Hill coefficient of 1.67 +
− 0.36, greater than 1.0 (P = 0.03). These
composite Hill coefficients were associated with EC50 values of
44 +
− 22 and 40 +
− 15 μM for ATP and ADP respectively. These
values indicate that adenine nucleotide binding to IRP-1 shows
strong positive co-operativity above 1 μM, and the binding does
not discriminate between ATP and ADP.
Binding to chemically prepared holo- and apo-protein
Figure 3 Characteristics of representative apo- and holo-protein
preparations
Holo- and apo-protein were prepared as described in the
Experimental section and were characterized by aconitase activity,
EMSA and Western blotting. Both holoprotein and rIRP-1 showed
low IRE binding that was substantially increased by treatment
with 2-mercaptoethanol (Figure 3A), which is thought to disrupt
(A) IRE-binding activity by EMSA of the starting material rIRP-1 and the modified proteins,
with or without incubation with 2 % (v/v) 2-mercaptoethanol (β-ME). (B) Western blot with an
anti-IRP-1 antibody of 0.5 μg of protein from each preparation, resolved by SDS/PAGE (8 %
gel). (C) Aconitase activity of rIRP-1, aporotein and holoenzyme, measured as described in the
Experimental section.
c The Authors Journal compilation c 2010 Biochemical Society
ATP binding to IRP-1
319
Table 1 Nucleotide-binding properties of apoprotein and holoprotein
preparations compared with the rIRP-1 starting material
Values are calculated by filter-binding and non-linear regression as described in the text and the
legend to Figure 2. Values represent the mean and range from two independent preparations.
ATP binding
ADP binding
Preparation
B max
Hill coefficient
B max
Hill coefficient
rIRP-1
Apoprotein
Holoprotein
2.13 +
− 0.17
2.65 +
− 0.35
1.64 +
− 0.13
1.67 +
− 0.08
2.50 +
− 0.46
1.83 +
− 0.04
2.10 +
− 0.07
3.37 +
− 0.24
1.94 +
− 0.44
1.74 +
− 0.06
2.07 +
− 0.01
1.80 +
− 0.01
the [4Fe–4S] cluster and convert aconitase into the binding form.
In contrast, the apoprotein preparation shows full binding activity.
On the other hand, production of the apoprotein completely
eliminates aconitase activity, whereas reconstitution leads to an
approximate 3-fold increase (Figure 3C).
Two independent preparations from a separate isolation of
rIRP-1 were used to study ATP and ADP binding (Table 1).
The new preparation of rIRP-1 showed Bmax values and Hill
slopes consistent with the above analyses, again indicating two
binding sites for both ATP and ADP, with co-operative binding.
These properties were retained for both nucleotides in the
holoprotein, with an indication of a possible additional site and
even stronger co-operativity in the apoprotein. However, higher K d
values suggested weaker binding, particularly with the apoprotein
(315 +
− 49 μM and 422 +
− 87 μM with the apoprotein for ATP and
ADP respectively; and 115 +
− 3 μM and 153 +
− 37 μM with the
holoprotein). These results indicate that, whereas both aconitase
and RNA-binding activity are present in a relatively low portion of
rIRP-1 as isolated, both properties can be restored. Furthermore,
both holo- and apo-protein appear to contribute to the co-operative
binding of nucleotides at two sites in the concentration range 0–
200 μM. Subsequent experiments with unmodified rIRP-1 are
justified in order to minimize possible conformational changes
caused by the chemical treatments used to isolate the holo- and
apo-protein species, as suggested by the higher K d values.
Hydrolysis is required for ATP-induced co-operative binding
Binding of label from [α-32 P]ATP, but not [γ -32 P]ATP, to IRP-1
suggests that hydrolysis is necessary for binding. To confirm
this, we attempted to suppress hydrolysis by performing the
binding reaction at 4 ◦C. However, neither label was bound under
these circumstances (Supplementary Figure S3A at http://www.
BiochemJ.org/bj/430/bj4300315add.htm). This could be because
of a loss of hydrolytic activity, if hydrolysis is indeed a requirement for binding, or because of a change in conformation
that disrupts the binding site. Therefore we measured binding
of [α-32 P]ADP (prepared by the reaction of [α-32 P]ATP with
hexokinase) directly. [α-32 P]ADP binding saturated in a time
similar to [α-32 P]ATP (compare Figure 1B and Supplementary
Figure S3B), but with a lower Bmax = 1.04 and a K d = 42.4 μM
(Supplementary Figure S3C). [α-32 P]ADP also fails to bind at 4 ◦C
(Supplementary Figure S3A). Taken together, these data suggest
that the primary binding site is indeed occupied by ADP, but
that ATP hydrolysis allows occupation of this site. Subsequent
experiments reported below are performed with [α-32 P]ATP.
The ATP analogue AMP-PNP, which is non-hydrolysable
between the β and γ phosphorus atoms, had no effect on IRE
binding either in cell extracts or with recombinant protein [11].
This further implies that ATP must be hydrolysed in order to
modulate the IRP–IRE interaction, but it remains unclear whether
Figure 4
IRP-1.
Influence of competing nucleotides on [α-32 P]ATP binding to
(A) IRP-1 (1 μg/20 μl) was incubated with 100 nM [α-32 P]ATP in the presence of 0.5–100 μM
unlabelled nucleotide (ATP, ADP, AMP-PNP, GTP or CTP) for 30 min at 25 ◦C. After
autoradiography of the nitrocellulose membrane, spots were cut and counted by liquid
scintillation. Values are means +
− S.D. from three separate experiments. (B) Bound c.p.m. from
the data in (A) are shown to underscore the requirement for ADP or a hydrolysable adenosine
nucleotide analogue to achieve binding. (C) Competition of [α-32 P]ATP binding. IRP-1 was
pre-incubated with 100 nM [α-32 P]ATP in the presence of 5 μM unlabelled ATP, and the bound
counts were taken as 100 %. The mixture was then incubated with the indicated concentration of
unlabelled nucleotide for 30 min at 25 ◦C. The x -axis shows the amount of unlabelled nucleotide
added after the pre-incubation.
the energy derived from hydrolysis is important, or rather the
product, ADP, is sufficient to inhibit binding of IRP-1 to IRE.
To address this, we incubated rIRP-1 with a non-hydrolysable
analogue, AMP-PNP, and measured binding of [α-32 P]ATP
in the filter-binding assay. In contrast with ATP and ADP,
AMP-PNP does not produce co-operative radioligand binding
to IRP-1 (Figure 4A). This further suggests that the product
ADP is required to effect a change in the IRP-1 conformation
that is unfavourable for IRP-1–IRE complex formation. CTP
and GTP also fail to show co-operativity with ATP binding
(Figure 4A). Absolute binding (c.p.m.) of [α-32 P]ATP is increased
approx. 10-fold by 1 μM unlabelled ATP or ADP, despite the
decreasing radiospecific activity with increasing addition of
non-radiolabelled nucleotide (Figure 4B). AMP-PNP, CTP and
GTP do not increase counts above background. However, when
[α-32 P]ATP was bound in the presence of 5 μM unlabelled
ATP (i.e. above the concentration effecting enhanced nucleotide
binding), and then excess unlabelled nucleotide was added
subsequently to compete with the label, the nucleotides ATP,
ADP, AMP-PNP and CTP were equally effective in competition.
We conclude that a conformational change brought about by
ADP renders the protein susceptible to competitive binding
by the other nucleotides. Surprisingly, GTP was highly
competitive under these circumstances, competing with ADPinduced binding at 5 μM (Figure 4C).
c The Authors Journal compilation c 2010 Biochemical Society
320
Z. Popovic and D. M. Templeton
Figure 6
CD spectra of IRP-1 in the presence of ATP/ADP
(A) Spectra are of IRP-1 recorded in the absence (most negative θ) of ATP or after titration with
5, 10, 20, 30, 40 or 50 μM (least negative θ) ATP. The inset shows the spectra of 0–50 μM ATP
alone, in the same buffer. (B) Spectra of two samples of IRP-1 are plotted (lower tracings), and
then ATP (50 μM) was added to one sample and ADP (50 μM) to the other. The spectra with
the nucleotides are superimposable (upper tracings).
Figure 5 Separation of ATP–IRP-1 complexes on polyacrylamide and
agarose gels
(A) Recombinant IRP-1 (1 μg) was incubated with 100 nM [α-32 P]ATP and 1 μM unlabelled
ATP for 30 min at 25 ◦C, and the mixture separated by SDS/PAGE. A silver stain of the gel
shows molecular-mass markers (lane M) and IRP-1 (lane Ag). The same gel was subjected
to autoradiography (lane R) and Western blotting with an anti-IRP-1 antibody (lane W). (B)
Recombinant IRP-1 (1 μg) was incubated with 100 nM [α-32 P]ATP in the presence of the
indicated concentrations of unlabelled ATP for 30 min at 25 ◦C. The mixture was then subjected
to non-denaturing agarose gel (0.8 %) electrophoresis, and the gel was dried and visualized by
autoradiography.
ATP and ADP binding change the IRP-1 structure
We previously demonstrated the binding of ATP to IRP-1 by
photolabelling with 8-azido-[α-32 P]ATP [11]. In the present study
we confirmed nucleotide binding after incubation of IRP-1 with
[α-32 P]ATP in the presence of unlabelled ATP and separation by
SDS/PAGE. Radioligand binds to the major band of rIRP-1 on
a silver-stained gel, as seen on autoradiography, and this band is
further identified as IRP-1 by Western blotting with an anti-IRP-1
antibody (Figure 5A).
The above radionucleotide-binding experiments indicate the
allosteric nature of ATP–IRP-1 interactions, and furthermore
indicate that ADP binding produces a state of IRP-1 that does
not bind IRE. The change in the binding state of IRP-1 occurs at
approx. 1 μM AT(D)P when binding of radionucleotide sharply
increases. We attempted to visualize the ATP-modified bound
form(s) by means of non-denaturing agarose gel electrophoresis.
Native electrophoresis indeed suggests changes of IRP-1 structure
(Figure 5B), in that an increase of non-radiolabelled ATP
concentration results in species that move slower into the agarose
gel, opposite to the expectation if the mobility differences were
due to increased negative charge on binding AT(D)P. As in the
filter-binding assay, bound [α-32 P]ATP is not competed out by
c The Authors Journal compilation c 2010 Biochemical Society
addition of unlabelled ATP up to 4–6 μM, consistent with cooperatively enhanced binding in the filter-binding assay. The shift
in electrophoretic mobility was already apparent at 0.5 μM ATP,
somewhat below the threshold concentration for enhanced ATP
binding, and plateaus at approx. 10 μM. No 32 P remained on the
gel when radioligand and non-radiolabelled ATP were incubated
with BSA, IRE or without protein (results not shown), confirming
specific binding to IRP-1.
Addition of SDS to the loading buffer prevented the apparent
conformational change and, irrespective of the concentration of
ATP, the IRP-1–ATP complex migrated at the same position
in the presence of SDS (results not shown). No complex of
radioligand with IRP-1 was detected when IRP-1 was incubated
with [γ -32 P]ATP, or with [α-32 P]ATP in the presence of excess
unlabelled AMP-PNP or CTP (results not shown). From the
previous experiments one would predict that ADP, too, would
induce a structural change of IRP-1, and this is indeed the case
(see Figure 8B, discussed below).
To determine that these shifts in agarose electrophoretic
mobility truly arose from a conformational change, we recorded
CD spectra of the protein in the absence and presence of ATP
(Figure 6A). The native protein shows a minimum at approx.
208 nm, consistent with an estimated α-helical content of approx.
25 % [28]. On titration with ATP, a progressive loss in optical
rotation, θ , between 208 and 230 nm is seen from 5 to 50 μM ATP.
In this concentration range, ATP does not contribute significantly
to the spectrum (Figure 6A, inset). Tracings of parallel samples
of IRP-1 treated with either ATP or ADP (each 50 μM) show
identical spectral shifts in the range 208–230 nm (Figure 6B).
Cytosolic aconitase activity requires an intact [4Fe–4S] cluster,
and is conformation- and temperature-sensitive, losing up to
30 % of enzyme activity after 16 h at 0 ◦C [29]. We measured
the aconitase activity of IRP-1 held on ice compared with
that incubated at 37 ◦C, a process that leads to loss of the
ATP binding to IRP-1
Figure 7
321
Effect of nucleotides on aconitase stability
Recombinant IRP-1 was held on ice (first bar) or incubated at 37 ◦C for 30 min in the absence
(second bar) or presence of the indicated nucleotide (5 mM) for 30 min. Aconitase activity was
then measured as the conversion of cis -aconitate as described in the Experimental section. Bars
marked * differ from incubation at 37 ◦C without nucleotide (second bar from left) at P < 0.01
(n = 3).
rather unstable aconitase activity. We then determined whether
inclusion of various nucleotides could stabilize aconitase activity.
In a typical experiment where 70 % of the control activity was
lost after 30 min of incubation, both ATP and ADP offered
partial protection (approx. 50–60 % of full activity, P < 0.01)
(Figure 7). No protection was achieved with CTP, GTP or the
non-hydrolysable AMP-PNP.
IRP-1(C437S) shows diminished ATP binding and hydrolysis
Mutation of Cys437 eliminates a ligand of the [4Fe–4S] cluster
and destabilizes cluster formation [30,31]. Wild-type IRP-1 and
IRP-1(C437S) purified on Ni-NTA show a similar pattern on
SDS/PAGE and Western blots (Figure 8A). As expected [30,31],
the mutant shows significantly greater IRE binding (Figure 9A)
and absent aconitase activity (results not shown). However, its
ATP binding is significantly diminished (Figure 9B); whereas
IRP-1 binds up to 4 mol of ATP (Bmax = 3.97, K d = 66 μM), the
mutant binds only 1 mol of ATP (Bmax = 1.12, K d = 123 μM).
Furthermore, the mutant fails to show co-operative adenosine
nucleotide binding (results not shown). ATP hydrolysis proceeds
at a lower rate with the mutant (V max = 75 +
− 17 nmol/min per mg
48
nmol/min
per mg of protein for
of protein compared with 147 +
−
wild-type), and at a K m of 0.18 +
− 0.09 mM for mutant compared
with 0.30 +
− 17 mM for wild-type (Figure 9C); however, 5 mM
ATP, but not GTP, is able to suppress IRE binding of both wildtype and mutant proteins (Figure 9D). Whereas wild-type protein
is shifted on non-denaturing agarose gels in the presence of both
ATP and ADP, such a structural change is not evident with the
mutant protein (Figure 8B).
DISCUSSION
In the present study we confirm that rIRP-1 binds ATP and ADP,
and upon binding undergoes structural changes that influence its
electrophoretic mobility and CD spectrum. At ATP concentrations
in the nanomolar range, IRP-1 has a low affinity for ATP that
remains up to approx. 1 μM ATP. Bound ATP is hydrolysed,
as demonstrated by failure to detect any significant [γ -32 P]ATP
binding. This can be explained by the ATPase activity of IRP-1
[11]. However, increasing ATP concentration to the micromolar
Figure 8 Analysis of nucleotide binding of wild-type IRP-1 and mutant
IRP-1(C437S)
(A) Wild type (wt) or mutant (m) IRP-1 were separated on SDS/PAGE and visualized by either
Coomassie Blue staining (5 μg of protein) or Western blotting (2.5 μg of protein) with an
anti-IRP-1 antibody, as indicated. The left-most lane (Mr) shows molecular-mass markers.
(B) Non-denaturing agarose gel electrophoresis was performed as described in Figure 6, with
either IRP-1 or IRP-1(C437S), after incubation with 100 nM [α-32 P]ATP in the presence of the
indicated concentrations of unlabelled ATP or ADP. An autoradiogram of the dried gel is shown.
range and above has a co-operative effect on IRP-1 binding.
Mg2+ is required for ATP binding, in agreement with previous
findings that Mg2+ is important for IRP-1 activation [32]. In
the micromolar ATP range, binding is strongly dependent on
protein concentration, a characteristic of allosteric proteins. Cooperativity is usually the result of conformational change, and
indeed addition of ATP/ADP results in an altered structure visible
as discrete forms of the IRP-1–ATP complex with decreased
mobility on native agarose gels, and a shift in the CD spectrum in
the 5–50 μM range. Co-operative binding permits a much more
sensitive response to nucleotide concentration.
Previously, we demonstrated that the IRP-1–IRE complex
is absent in the presence of ATP [11]. Inhibition of complex
formation can be achieved with either ATP or ADP, suggesting that
the nucleotides stabilize the protein in a non-IRE-binding form.
This effect was not observed with non-hydrolysable analogues
[11], and again in the present study the non-hydrolysable analogue
AMP-PNP does not bind to IRP-1; and it does not induce a cooperative effect, does not compete with ATP/ADP (Figure 4B) and
does not form shifted complexes on native agarose gels (results
not shown). Nucleotides may stabilize non-IRE-binding form(s),
and ATP and ADP provide at least temporary protection against
loss of aconitase activity upon incubation at 37 ◦C. However, cooperative binding of the hydroysable nucleotides is conserved in
both the chemically prepared holo- and apo- forms of the protein,
and in the apoprotein there is a tendency for increased binding and
co-operativity. Thus nucleotide binding may stabilize both states
of the protein independent of a switch between them.
c The Authors Journal compilation c 2010 Biochemical Society
322
Figure 9
Z. Popovic and D. M. Templeton
Comparison of IRP-1 and IRP-1(C437S)
(A) Filter-binding assay of wild-type (䊊) and mutant (䊉) proteins (0.5 μg/20 μl) incubated with 2.5–100 ng radiolabelled IRE for 30 min at 25 ◦C. The inset shows a representative autoradiogram.
Spots were cut for scintillation counting. Values are means +
− S.D. from triplicate wells of duplicate experiments. (B) Filter-binding assay of wild-type (䊊) and mutant (䊉) proteins (1 μg/20 μl)
incubated with 50 nM [α-32 P]ATP in the presence of increasing concentrations of unlabeled ATP for 30 min at 25 ◦C. K d and B max values calculated from non-linear regression are as reported in
the text. (C) ATPase activity of wild-type (䊊) and mutant (䊉) proteins. Protein (1.25 μg/50 μl) was incubated with 0–2 mM ATP for 30 min at 37 ◦C and free Pi was detected with Malachite Green
at 660 nm. K m and V max values calculated from non-linear regression are as reported in the text. (D) EMSA of wild-type (w) and mutant (m) proteins with radiolabelled IRE, in the presence of the
indicated concentration of ATP or GTP.
It has been estimated that only 1–5 % of IRP-1 is in the RNAbinding form in tissues [6,33,34]. Consistent with this, we found
that purification of IRP-1 on an IRE-affinity column gave a
relatively low yield of protein with a V max for ATP hydrolysis
of only 3.4 nmol/min per mg of protein [11]. In contrast, total
rIRP-1 purified on Ni-NTA is found in the present study to
have a V max 40-fold higher (147 nmol/min per mg of protein).
This indicates that the IRE-binding form is a relatively minor
component, and is consistent with a large increase in binding
upon preparation of the apo-form (Figure 3A). The C437S mutant
strengthens the argument that IRE binding is associated with an
aconitase-deficient state, whereas ATP binding and hydrolytic
activity are greater in the non-IRE-binding state. Increased IREbinding activity of the mutant is associated with a V max for
ATP hydrolysis only half that of wild-type protein (75 nmol/min
per mg of protein). Loss of one nucleotide-binding site in the
mutant protein, but not the apoprotein preparation, indicates
additional structural changes in the mutant beyond those due to
the absence of the [4Fe–4S] cluster. Despite retention of only a
single site, ATP binding is still able to block interaction with IRE
(Figure 9D).
Strong competition by GTP with ATP binding occurs only in
the protein structure that shows enhanced binding at higher ADP
concentration. In this context, it is interesting to note that ATP
and GTP are required for efficient biogenesis of the [4Fe–4S]
cluster of mitochondrial aconitase [35,36], and a possible role of
the structurally altered form of cytosolic aconitase in responding
to GTP levels is intriguing.
Values of K d found for ATP by filter binding (54.8 +
− 12.5 μM)
and to protein bound on Ni-NTA agarose (74.4 +
49.8
μM) are
−
consistent with that reported previously for IRE-purified protein
(86 +
− 17 μM) [11], and found in the present study for ADP
(121 +
− 30 μM). Total cellular ATP levels of approx. 5 mM [37]
imply that IRP-1 would be saturated with ADP under normal
c The Authors Journal compilation c 2010 Biochemical Society
circumstances, and stabilized in a non-IRE-binding aconitaseactive form. If this is true, it could account for the studies noted
above showing that only a small portion of cellular IRP-1 is in
the IRE-binding form [6,33,34], and the evidence from IRP-1gene-ablated mice that IRP-2, and not IRP-1, is responsible for
post-transcriptional regulation of iron homoeostasis [34].
IRP-1 lacking the [4Fe–4S] cluster is degraded through
ubiquitin ligase by the same mechanism as IRP-2 [38,39]; in ironreplete cells the [4Fe–4S] form should be stable. If a pool of apoIRP-1 exists, it should be protected (e.g. by oligomerization or
compartmentalization), possibly through ATP-induced conformational changes. Cytosolic aconitases from both bacteria and
mammals have a tendency to oligomerize. E. coli aconitase
AcnB forms a homodimer both in vitro and in vivo, and the
monomer–dimer transition is dependent on the availability of
iron [40]. Only the monomer can act as a post-transcriptional
regulator [40] binding IRE. Yikilmaz et al. [28] found that purified
human recombinant apo-IRP1 exists in a slowly reversible
monomer–dimer self-association equilibrium. They showed that
binding of IRE drives a conformational change toward a complex
in which IRP1 is entirely monomeric.
Interactions of IRP-1 with ATP/ADP occur in two nucleotide
concentration regimes. On the one hand, concentrations in the
1–10 mM range block RNA binding [11] and stabilize aconitase
activity (Figure 7). On the other hand, concentrations of
1–10 μM produce a shift in conformation dectectable by CD and
native agarose gel electrophoresis, and encompass a transition
reflecting positive co-operativity in adenosine nucleotide binding.
Typical cellular ATP levels at approx. 5 mM [37,41] imply a role
in regulation of the IRE-binding/aconitase transition. However,
the binding constants of AT(D)P ([11] and the present study)
imply saturation of binding sites at this concentration, which may
explain why very little IRP-1 is in the IRE-binding form and
may function preferentially as a true cytosolic aconitase.
ATP binding to IRP-1
In the lower concentration range, it is unclear how micromolar
nucleotide levels could effect functional changes in the comparatively ATP-rich environment of the cell. Nevertheless, it
is true that many biological phenomena are regulated in this
concentration region. Documented effects of ATP on actin
polymerization are reported to have a half-maximal effective
concentration (EC50 ) of 200 nM in U937 cells, and plateau at
100 μM [42]. The EC50 for stimulation of ERK (extracellularsignal-regulated kinase) phosphorylation leading to proliferation
in cultured smooth muscle cells is 2–3 μM ATP [43]. The
K m values of many ATPases are in the micromolar range,
suggesting again that regulation at this level is relevant. Lower
local concentrations of ATP/ADP may occur, e.g. due to
compartmentalization. At least 50 % of cytosolic ADP is proteinbound [44]. Thus whether IRP-1 experiences sub-saturating
cytosolic ATP concentrations in the cell remains an open question.
Certainly our estimated K m value of 5.3 μM [11] is in keeping with
K m values reported for other ATP-hydrolysing proteins, e.g. E. coli
DnaK (20 μM) [45], Hsc70 (heat-shock cognate 70 stress protein)
(1.4 μM) [46] and F1-ATPase (15 μM) [47]. Furthermore, these
proteins have V max values in the range 1.1–3.5 nmol/min per mg
of protein, comparable with the value of 3.4 nmol/min
per mg of protein reported for IRP-1 [11], supporting a physiological relevance to the observed high-affinity ATP binding.
Finally, high ATP concentrations that suppress the IRE–IRP-1
complex would favour ferritin translation. Concomitantly, high
ATP inhibits oxidative phosphorylation [37], and the newly
synthesized ferritin would be available to shelter excess O2 and
iron [48]. In a period of high cytosolic ATP and attenuated
oxidative phosphorylation, surplus citrate from the tricarboxylic
acid cycle is exported from mitochondria and utilized for lipid,
glutamate and NADPH production. It has been suggested that
cytosolic aconitase is involved in these metabolic processes
[5,49,50].
AUTHOR CONTRIBUTION
Zvezdana Popovic and Douglas Templeton contributed to the experimental design, data
interpretation and to the writing of the paper.
FUNDING
This work was supported by the Canadian Institutes of Health Research [grant number
MT11270 (to D. M. T.)].
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Received 18 January 2010/9 June 2010; accepted 23 June 2010
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Biochem. J. (2010) 430, 315–324 (Printed in Great Britain)
doi:10.1042/BJ20100111
SUPPLEMENTARY ONLINE DATA
Interaction of iron regulatory protein-1 (IRP-1) with ATP/ADP maintains a
non-IRE-binding state
Zvezdana POPOVIC and Douglas M. TEMPLETON1
Laboratory Medicine and Pathobiology, University of Toronto, 1 King’s College Circle, Toronto, ON, Canada M5S 1A8
Figure S1
Binding and hydrolysis of ATP by IRP-1
(A) pH-dependence of ATP binding. IRP-1 (1 μg) was incubated with 100 nM [α-32 P]ATP in the
presence of unlabelled ATP (䊏, 10 μM; 䊉 100 μM) at the pH indicated. (B) Specificity of
the ATP–IRP-1 interaction. ATP binds to His–IRP-1 Ni-NTA agarose. IRP-1 (5 μg) was incubated
with 100 nM [α-32 P]ATP and 0–500 μM unlabelled ATP for 30 min at 25 ◦C following incubation with Ni-NTA agarose beads and subsequent elution of His-tagged IRP-1 as described in
the Experimental section of the main text.
1
Figure S2
Effect of ADP on binding of [α-32 P]ATP to IRP-1
(A) Representative filter-binding assay of 50 nM [α-32 P]ATP binding to IRP-1 (1 μg in a total
reaction volume of 20 μl) in the presence of the indicated concentrations of unlabelled ADP.
(B) Non-linear regression of liquid scintillation data from three independent experiments such
2
+
as shown in (A), giving an apparent B max = 2.37 +
− 0.30, K d = 1212− 30 μM (R = 0.89).
(C) Hill plot of the data from (B), giving a Hill slope of 1.39 +
− 0.35 (R = 0.88).
To whom correspondence should be addressed (email [email protected]).
c The Authors Journal compilation c 2010 Biochemical Society
Z. Popovic and D. M. Templeton
Figure S3
Binding of [α-32 P]ADP/ATP to IRP-1
(A) Filter-binding assays were performed with either 10 μM unlabelled ATP or ADP, in
the presence of 50 nM label (䊉 and 䊊, [α-32 P]ATP; 䊏 and 䊐, [α-32 P]ADP) for up to
60 min, at both 37 ◦C (䊉 and 䊏) and 4 ◦C (䊊 and 䊐). Values are means +
− S.D. of three
measurements at each time point. (B) Time course of binding of [α-32 P]ADP (50 nM) in the
presence of 10 μM unlabelled ADP, at room temperature (compare with Figure 1B in the main
text). (C) Non-linear regression of filter-binding data of [α-32 P]ADP giving B max = 1.04 and
K d = 42.4 μM (R 2 = 0.95). Values are means +
− S.D. from two experiments with four and five
replicates respectively.
Received 18 January 2010/9 June 2010; accepted 23 June 2010
Published as BJ Immediate Publication 23 June 2010, doi:10.1042/BJ20100111
c The Authors Journal compilation c 2010 Biochemical Society