From Unité Propre de Recherche 9002 du CNRS, Institut de Biologie Moléculaire et Cellulaire, 15 rue René Descartes, 67084 Strasbourg Cedex, France and the ¶ Gene Expression Programme, European Molecular Biology Laboratory, Meyerhofstrasse 1, D-69117 Heidelberg, Germany
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ABSTRACT |
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Human iron regulatory protein-1 (IRP-1) is a
bifunctional protein that regulates iron metabolism by binding to
mRNAs encoding proteins involved in iron uptake, storage, and
utilization. Intracellular iron accumulation regulates IRP-1 function
by promoting the assembly of an iron-sulfur cluster, conferring
aconitase activity to IRP-1, and hindering RNA binding. Using protein
footprinting, we have studied the structure of the two functional forms
of IRP-1 and have mapped the surface of the iron-responsive element
(IRE) binding site. Binding of the ferritin IRE or of the minimal
regulatory region of transferrin receptor mRNA induced strong
protections against proteolysis in the region spanning amino acids 80 to 187, which are located in the putative cleft thought to be involved in RNA binding. In addition, IRE-induced protections were also found in
the C-terminal domain at Arg-721 and Arg-728. These data implicate a
bipartite IRE binding site located in the putative cleft of IRP-1. The
aconitase form of IRP-1 adopts a more compact structure because strong
reductions of cleavage were detected in two defined areas encompassing
residues 149 to 187 and 721 to 735. Thus both ligands of apo-IRP-1, the
IRE and the 4Fe-4S cluster, induce distinct but overlapping alterations
in protease accessibility. These data provide evidences for structural
changes in IRP-1 upon cluster formation that affect the accessibility of residues constituting the RNA binding site.
Intracellular iron homeostasis in mammals and many other
eukaryotes is controlled at the post-transcriptional level by two related cytoplasmic proteins, the iron regulatory proteins (IRP-1 and
IRP-2)1 (for reviews see
Refs. 1 and 2). These two proteins bind to a conserved RNA stem-loop
structure, the so-called iron-responsive element. An increasing number
of mRNAs have been shown to carry one IRE motif in the
5'-untranslated regions of mRNAs encoding ferritin (L-
and H-chains), erythroid 5-aminolevulinate synthase, mitochondrial
aconitase, and succinate dehydrogenase, as well as in the
3'-untranslated region of transferrin receptor (TfR) and Nramp2
mRNAs, thus modulating their expression according to iron
availability (3-12). For instance, the interaction of IRP with a
single IRE in the 5'-untranslated region of ferritin mRNA inhibits
translation by preventing ribosome binding (13-15). On the other hand,
IRP binding to the five IREs present in the 3'-untranslated region of
TfR mRNA stabilizes the message against degradation (10, 16).
Therefore, the specific interaction between IRP and mRNA ensures a
coordinated regulation of the expression of proteins involved in the
uptake (TfR), storage (ferritin), and utilization (erythroid
5-aminolevulinate synthase) of iron (2). Because the IRPs appear to
regulate two enzymes of the citric acid cycle, they may also play an
important role in mediating iron regulation of mitochondrial energy
production (9, 10).
IRP-1 and IRP-2 are affected by iron in different manners. Whereas iron
increases the rate of degradation of IRP-2 (17, 18), it inhibits the
ability of IRP-1 to bind to IREs through formation of an iron-sulfur
cluster. Interestingly, the 4Fe-4S cluster assembly confers aconitase
activity to IRP-1 (19-24). IRP-1 can therefore directly sense and
respond to perturbations of iron levels and redox potential. Moreover,
phosphorylation of IRP-1 may also be involved in the regulation of its
two functions because it preferentially occurs in the apoprotein (25,
26). Unlike other enzymes that recognize mRNA (1), the functional
implication of the cytoplasmic aconitase activity of IRP-1 is not well
understood. However, in addition to the functional similarities,
sequence homologies between IRP-1 and the mitochondrial aconitase
suggest that these proteins may adopt similar structures (23, 27). In
particular, a striking level of conservation exists for the amino acids
located in the active site of mitochondrial aconitase. The crystal
structure of mitochondrial aconitase has revealed that domains 1-3 are
connected by a linker region to domain 4 (28), the iron-sulfur cluster
being located in a cleft formed between domains 1 and 3 and domain 4. Furthermore, mutational analysis (29, 30) and cross-linking data
(31-33) have indicated that multiple contacts occur between IRP-1 and
the IRE hairpin on both sides of the putative cleft. It is interesting
to note that some arginine residues required for the aconitase activity are also critical for IRE binding (29, 30). These data suggest that the
RNA binding site and the catalytic center are overlapping and provide a
rational explanation for the mutually exclusive functions of IRP-1.
Based on the crystallographic structure of mitochondrial aconitase, it
was also proposed that significant rearrangement of the IRP-1 may occur
to permit the access of the IRE (for a review see Ref. 34).
In the present paper, we used a protein footprinting approach to
compare the structure of human IRP-1 in its two functional states, the
RNA-binding form (apoprotein) and the aconitase form (holoprotein). The
results demonstrated structural changes in IRP-1 as a function of the
presence or the absence of the iron-sulfur cluster. We also identified
single amino acids of IRP-1 that are protected by either the minimal
regulatory region of TfR mRNA (which contains three IREs) or of the
ferritin IRE upon binding that are located in two defined distant
regions of IRP-1 on both sides of the putative cleft. Interestingly,
these two regions also become less accessible toward proteases in the
holoprotein. Together our data provide direct experimental evidence for
the structural rearrangements of IRP-1 that are assumed in current models (for a review see Ref. 2).
RNAs and IRP-1 Preparations--
TfR (TRS-G165) and ferritin
mRNAs were synthesized by in vitro transcription with T7
RNA polymerase from plasmids linearized with BamHI. TfR
mRNA was transcribed with T7 RNA polymerase from polymerase chain
reaction-generated DNA templates (35). RNAs were 5'-end-labeled with
[
Recombinant human IRP-1 was purified from the Escherichia
coli overproducing strain K12 pQE9-his-hIRF. The protein was
isolated by affinity chromatography on Ni2+-NTA beads
(Qiagen) as described previously (37). The protein was eluted with 50 mM imidazole and dialyzed against 20 mM
Tris-HCl, pH 7.5, 1.5 mM MgCl2, 40 mM KCl, 5 mM 2-mercaptoethanol (Buffer A). The
protein was then purified on a Mono-Q column equilibrated in Buffer A
and eluted from the column with a salt gradient (40 mM to 1 M KCl). IRP-1 was then concentrated using a Centricon 30K
(Amicon), washed several times in 20 mM Tris-HCl, pH 7.5, 1.5 mM MgCl2, 5 mM
2-mercaptoethanol, 5% glycerol (Buffer B), and stored at
Reconstitution of the [4Fe-4S] cluster in the affinity-purified human
IRP-1 (400 µg) was performed in 250 µl of buffer containing 50 mM Hepes-NaOH, pH 7.6, 150 mM potassium
acetate, 1.5 mM MgCl2, 5% glycerol (Buffer N)
in the presence of 3 mM FeSO4, 6 mM
Na2S for 15 min at 37 °C (38). The cytoplasmic aconitase
form of IRP-1 was purified on a TSK2000 gel filtration column (fast
protein liquid chromatography) equilibrated in Buffer B. Then the
protein was concentrated using a Centricon 30K. The aconitase activity was determined with the method of Rose and O'Connell (39) by following
the formation of NADPH at 340 nm as a function of time in a coupled
aconitase/isocitrate dehydrogenase reaction. The reconstituted
aconitase activity was found to be 40 units/mg.
Gel Retardation Assays--
Binding reactions containing
5'-end-labeled IRE and increasing concentrations of either IRP-1 or of
the aconitase form of IRP-1 were incubated at 4 °C for 20 min in
buffer N. RNA-protein complex formation was analyzed by a 8%
non-denaturing polyacrylamide gel as described previously (40).
Protein Footprinting--
IRP-1 protein or its aconitase form (4 µM) either alone or in the presence of RNA (TRS-G165 or
IRE) were subjected to partial proteolytic digestions using different
proteinases (Roche Molecular Biochemicals). Recombinant IRP-1 was first
treated with 2% 2-mercaptoethanol. Then, RNA binding was assayed in
the presence of a 2-fold excess of TRS-1 or ferritin IRE at 4 °C for
20 min in 50 mM Hepes-NaOH, pH 7.6, 150 mM
potassium acetate, 1.5 mM MgCl2, and 5 mM 2-mercaptoethanol. Proteolytic digestions were performed
in 10 µl with Arg-C (0.02 unit), Asp-N (0.004 µg), Glu-C (0.1 µg), Lys-C (0.02 U), or trypsin (0.2 µg) at 37 °C for 30 min.
The reactions were stopped by the addition of 0.5 volume of 2× sample
buffer containing 50 mM Tris-HCl, pH 6.8, 8% SDS, 25%
glycerol, 4% 2-mercaptoethanol, 0.02% bromphenol blue. The samples
were denatured at 95 °C for 2 min before running on 5%
stacking/15% separating polyacrylamide-SDS gels. To get optimal
resolution of small peptides, the cleavage products were resolved using
10% stacking/16.5% separation polyacrylamide-Tricine-SDS gel
electrophoresis. Prestained rainbow proteins (from 90 kDa to 7.7 kDa)
were used as internal markers (Bio-Rad). These experiments were
repeated five times with different protein purifications. The main
cleavages were reproducibly found in all of the experiments. However,
the intensity of Asp-N specific cleavages varied in some of the
experiments depending on the batch of protease used.
Analysis of the Proteolytic Cleavages--
After
electrophoresis, the generated peptides were transferred from SDS gels
to polyvinylidene difluoride membranes (Hybond-P, Amersham Pharmacia
Biotech). The transfer was performed at 1 mA/cm2 in 25 mM Tris-HCl, pH 8.3, 0.2 M glycine, and 20%
methanol for 1 h at 20 °C using a semi-dry blotting apparatus.
The membrane was then blocked for 15 min at 20 °C in 20 mM Tris-HCl, pH 7.6, 137 mM NaCl, 0.1% Nonidet
P-40 (TBST buffer) with 10% milk powder. After washing three times in
TBST buffer, the membrane was incubated at 4 °C for at least 2 h with a 1:3300 dilution of RGS-His mouse antibody (Qiagen) in TBS
buffer. After three washes with TBST, the membrane was incubated for
1 h at 20 °C with 1:1000 dilution of alkaline
phosphatase-conjugated secondary antibody (anti-mouse FAB/POD, Roche
Molecular Biochemicals). After three washing steps with buffer TBST,
the detection was done using an ECL Western blotting kit (Amersham
Pharmacia Biotech). The bands were then revealed after autoradiography
of the membrane (5 s to 1 min).
Assignment of the Proteolytic Cleavages in IRP-1--
We probed
the accessibility of recombinant human IRP-1 (37) using a set of
endoproteases that cleave after basic and acidic residues. The
accessibility of lysine residues was probed with Lys-C and trypsin,
arginines with Arg-C and trypsin, aspartic acid with Asp-N and Glu-C,
and glutamic acid with Glu-C. These proteinases were used under native
conditions that are optimal for the stability of the RNA-IRP-1
complexes. We also verified that none of these proteinases contain
RNase activity. Cleavage conditions (time scale and concentration
dependences) were adjusted so that less than 50% of the input protein
was cleaved and thus that multiple cleavage of the protein was
unlikely. All the cuts that were considered appeared with similar
kinetics, at low protease concentration, and were reproducibly found.
Under these conditions of limited proteolysis, we favor detection of
only primary cleavages that reflect the accessibility of amino acids
within the native protein structure. We used a strategy derived from
Heyduk and Heyduk (41), which allows detection of the cleaved peptide
by immunostaining with antibodies specific to the N or C terminus of
protein. In our study, a recombinant IRP-1 protein carrying a
RGS-His-tag at its N terminus was expressed in E. coli and
purified by affinity chromatography (37). It was previously shown that recombinant IRP-1 efficiently binds to the ferritin IRE and can be
converted to an aconitase in vitro (37). This implies that the His-tag at the N terminus of IRP-1 does not interfere with IRE
binding and Fe-S cluster formation, respectively. After limited proteolysis of IRP-1, the resulting peptides were separated according to size on SDS-polyacrylamide gel electrophoresis (PAGE) and then electroblotted to a polyvinylidene difluoride membrane. The full-length protein and the RGS-His-tag peptides were revealed by using antibodies specific to the RGS-His-tag (Fig.
1A). The mobility of the bands on SDS-PAGE is directly proportional to the distance of the cleavage site from the N terminus of the protein. Thus, by comparing the electrophoretic mobility of appropriate protein standards and the
cleavage products generated by proteinases with different amino acid
specificities, identification of the cleavage sites can be
accomplished. The positions of the cuts were determined by plotting the
logarithm of the molecular masses of the assigned fragments against the
migration distances in the gel because an almost linear correlation for
polypeptide masses above 5 kDa has been described (41, 42). An example
shown in Fig. 1B revealed that the experimental values are
consistent with theoretical predictions and that the gel resolution
allows assignments at the level of single amino acids. No information
was obtained for peptides shorter than 8 kDa due to the low efficiency
of transfer of small peptides onto polyvinylidene difluoride membranes.
The experiments have been repeated five times with three different
preparations of IRP-1 and showed reproducible proteolytic cleavages.
All the cleavage sites in IRP-1 that were identified consistently in
all the experiments are listed in Table
I.
A large part of IRP-1 is not sensitive to proteolysis, consistent with
previous results (25), especially the region encompassing residues 190 to 380. The proteases used here cleave residues that are exposed to the
surface of IRP-1 and hence to the solvent because they are sensitive to
steric hindrance due to their relatively large molecular sizes (23.5 kDa for trypsin, 27 kDa for Glu-C and Asp-N, 33 kDa for Lys-C, and 59 kDa for Arg-C). Most of the cleavages are located in three defined
areas comprising residues 75 to 160, 375 to 400, and 720 to 755. In the
N-terminal domain, several fragments with molecular masses around 19 kDa were produced by all the proteases. Taking into account the
relative mobility of these bands and specific markers, the cleavage
specificity of the respective proteases and the potential target in
IRP-1 sequence, it was possible to assign the cleavage sites: two
trypsin- and Arg-C-specific bands were assigned to Arg-149 and Arg-135 (Fig. 1). The strong Asp-N-specific cleavage most likely occurred before Asp-137 because the band migrated at the same position as the
trypsin- and Arg-C-specific band at Arg-135. The Lys-C- and
trypsin-specific band that migrated between Arg-149 and Arg-135, was
attributed to Lys-141. Glu-C induces two main cleavages in this area,
one site occurs just below the band corresponding to Arg-149 and was
identified as Glu-148. The distance between the two Glu-C-specific
bands favors position Glu-155 as the potential second cleavage site
(Fig. 1, A and B). Using the same procedure, the
smaller fragments generated by Asp-N (Asp-87, Asp-102, Asp-125), Lys-C
(Lys-79, Lys-105), Arg-C (Arg-82), and trypsin (Lys-79, Arg-82,
Arg-101, Lys-105) were identified after short electrophoretic migrations on SDS-Tricine gels. Conversely, the strong cleavages induced by trypsin, Arg-C, and Glu-C in the C-terminal domain of IRP-1
were assigned on long run SDS-PAGE gels. The major Glu-C-specific cut
migrated with a molecular mass around 70 kDa (Fig. 1) and was assigned
by sequencing the corresponding large and small fragments using
automated Edman degradation (results not shown). The large fragment
corresponded to the N terminus of IRP-1, and the N-terminal sequence of
the 30-kDa fragment was SWNALA, indicating that the major
Glu-C-specific cut occurred at Glu-621. This cut was also previously
reported (20). We used the same strategy to identify the three strong
Arg-C-specific cleavages in the N-terminal domain. The resulting
fragments migrate slower than fragment Glu-621, suggesting a cleavage
at Arg-721, Arg-728, and Arg-732. This was again confirmed by
sequencing the two major corresponding C-terminal fragments using Edman
degradation, which gave the sequences GTFA and LLNRFL.
Probing the IRE Binding Site on IRP-1--
To identify structural
changes of IRP-1 induced by IRE binding, we used the footprinting
approach to map the IRE binding site in IRP-1. This also provided
information on potential amino acids involved in the RNA binding (42,
43). The RNA-induced effects on IRP-1 structure were compared using as
RNAs the IRE of the ferritin mRNA and the regulatory region of the
human TfR mRNA, which contains three of the IRE elements (TRS-G165)
(see Fig. 2C). IRP-1 was
probed with several proteases (trypsin, Arg-C, Lys-C, Asp-N, and Glu-C)
in the presence of a 2-fold excess of the RNA. Under the experimental
conditions used, the protein was quantitatively bound to the RNAs
(results not shown). The results are shown in Figs. 1 and 2 and are
summarized in Table I. The data show that the IRE of the ferritin
mRNA induced protections against several proteases in two defined
regions of IRP-1, located in the two terminal domains. In the
N-terminal domain, total protection was observed at Lys-79, Arg-82,
Asp-87, Arg-101/Asp-102, Lys-105, Lys-106, Asp-125, Glu-155, and
Arg-187. This was accompanied by significant increased cleavages at
Arg-134, Arg-135, Asp-137, and Lys-141 whereas the Glu-C-specific cut
at Glu-148 remained unchanged (Figs. 1A and 2, Table I). One
discrepancy was observed at Arg-149, which was strongly protected by
the IRE hairpin toward Arg-C and was still accessible toward trypsin.
This result may be explained by the fact that Arg-C has a higher
molecular weight and therefore is more sensitive to steric hindrance
(Figs. 1A and 2). Other significant protections were
observed in the C-terminal domain at Arg-721 and to a lesser extent at
Arg-728 and Arg-732 upon IRE binding (Table I). Binding of the minimal
regulatory region of TfR mRNA (TRS-G165) induced the same changes
in IRP-1 as IRE of the ferritin mRNA (Fig. 2B),
indicating that binding of TRS-G165 RNA is restricted to the IRE motif.
The addition of the same amount of tRNA did not alter the cleavage
pattern, demonstrating that the changes in IRP-1 induced by IRE or
TRS-G165 binding were specific.
Structural Changes of IRP-1 Induced by Iron-Sulfur Cluster
Assembly--
It was previously proposed that the aconitase form of
IRP-1 may display structural differences compared with apo-IRP-1
because the holoprotein does not efficiently bind to IREs (for a
review, see Ref. 34). A recent study indeed showed that the holoprotein appears to be more resistant than the RNA binding form of IRP-1 toward
limited proteolysis (25). Therefore, we mapped the structural changes
that occur in IRP-1 depending on the iron-sulfur cluster status. It was
previously shown that the iron-induced reduction of IRE binding
activity in cells could be reconstituted by treatment of IRP-1 with an
excess of FeSO4 in the presence of cysteine (37). First we
have verified that reconstitution of the iron-sulfur cluster in IRP-1
conferred aconitase activity to the protein using a coupled
aconitase/isocitrate dehydrogenase reaction. Furthermore, we also
showed that the holoprotein did not bind to IRE hairpin using
non-denaturing gel electrophoresis (data not shown). This is further
supported by the fact that ferritin IRE or TRS-G165 RNAs do not affect
the proteolytic cleavage pattern of the aconitase form (Fig.
2B). The holoprotein was then subjected to probing by
proteases in the presence or absence of the ferritin IRE or TRS-G165
RNA (Fig. 2). These experiments were repeated three times with three
different preparations of the aconitase form of IRP-1. Several
reproducible protections induced by Fe-S cluster formation were
detected in two distant regions, indicating that the holoprotein was
more resistant than the IRE binding form of IRP-1 toward proteases, consistent with previous findings (25). Strong protections occurred in
the N-terminal domain at Arg-149 toward Arg-C, at Glu-155 toward Glu-C,
and at Arg-187 toward trypsin, and in the C-terminal domain at Arg-721,
Arg-728, Arg-732 toward trypsin and Arg-C, at Lys-736 toward Lys-C, and
at Asp-751 toward Asp-N (Fig. 2, Table I). The protected residues were
located in two regions far away from the [Fe-S] cluster coordination
sites and most likely reflect conformational changes of IRP-1.
Interestingly all these residues were also found protected in IRP-1
upon IRE binding. These data suggest that [Fe-S] cluster formation
induces conformational changes of IRP-1 that mask the IRE binding site.
For the present work, we used protein footprinting to follow the
structural changes of IRP-1 in different functional states, the
RNA-binding form (apoprotein), and the aconitase form (holoprotein). Five different proteinases were used to probe the IRP-1 structure under
native conditions. The peptide cleavage products were subsequently resolved on SDS-PAGE gels and readily identified by comparing the
different protease digestions and the relative mobility of the
generated peptides using appropriate internal controls (Fig. 1,
A and B). Our data first demonstrated that
binding of the minimal regulatory region of TfR mRNA (TRS-G165) or
of the ferritin IRE induces the same protections in IRP-1, indicating
that binding of TRS-G165 is restricted to the IRE hairpin. The RNA
effect was restricted to the N-terminal domain of IRP-1 encompassing
residues 80 to 187 and to the region 721 to 732 at several arginine
residues. In the N-terminal domain, two sets of protected residues were found separated by several amino acids which were still accessible to
proteases (Fig. 1C). The protected residues are located in a
region that is highly conserved in IRPs. However an insertion of 73 amino acids in this domain found only in IRP-2 confers iron-mediated degradation (17). Interestingly, a mutant IRP-1 containing this insertion domain of IRP-2 bound RNA but prevented aconitase activity (44). It is interesting to note that this domain has been inserted in a
region that is still accessible toward proteases upon RNA binding (Fig.
1C). Some of these cuts were even enhanced upon IRE binding
indicating that conformational adjustments of IRP-1 may occur. The
N-terminal domain of IRP-1 is also of particular interest because
cross-links were identified between IRP-1 and residues 116 and 151 (31-33), and one UV-cross-linked site was postulated at Ser-127 (33).
The protected residue Asp-125 lies exactly in this area. This region is
highly conserved among IRPs (Fig. 1C, see Refs. 45 and
52-57) and contained two conserved residues, Asp-125 and His-126,
essential for the aconitase activity (29, 45). Therefore, it is
tempting to propose that the protections reflect direct contacts
between the IRE and IRP-1, masking the catalytic site. Interestingly,
one of the phosphorylation sites was located at Ser-138 (25) close to
the RNA binding site but in a region that remains accessible to
proteases (Fig. 1C). This may explain why phosphorylation
occurs preferentially in the apoprotein. Other regions have also been
shown to contribute to RNA binding. Cross-links were identified in a
region encompassing residues 480 to 623 (31), and the deletion of the
last 132 amino acids of IRP-1 results in a loss of IRE binding (20). It
is of interest that significant protections were detected at Arg-721
and to a lesser extent at Arg-728 and Arg-732. Interestingly, the
second phosphorylation site was detected in the same area at Ser-711 (25). These protections are also located in a region where an insertion
occurs relative to the sequence of mitochondrial aconitase (23, 27)
whereas this region is highly conserved in other IRPs (Fig.
1C, see Refs. 45 and 52-57). Also, Arg-699 participates directly in the active center of aconitase because it binds citrate, showing again the close proximity of the two functional sites (29).
Based on sequence alignments between mitochondrial aconitase and IRP-1,
we represented all the changes induced either by RNA or by iron-sulfur
binding on the tertiary structure of mitochondrial aconitase (Fig.
3). Interestingly, most of these
protected residues map in the putative cleft across domains 1 to 3 and
in domain 4. We and others previously showed that IRP-1 covers one
helical turn of the IRE and makes specific contacts with conserved
bases in the hairpin loop, at the bulged cytosine, and also with the ribose-phosphate backbone of the IRE (Fig. 2C and Refs. 35
and 46-50). Docking the IRE hairpin structure on the mt-aconitase
structure suggests that the size of the protected regions in IRE RNA is compatible with its localization deep in the putative cleft of IRP-1
covering residues 80-135 and 150-170 in the N-terminal domains and
residues 720-740 in the C-terminal domain, which are protected against
protease hydrolysis. However, the cleft as found in the crystal
structure of mt-aconitase is not open sufficiently to accommodate the
IRE hairpin and therefore conformational changes of IRP-1 are certainly
required to accommodate the RNA. It was proposed that the apoprotein
may adopt a more flexible conformation and that domains 1-3 and 4 may
be separated enough via the hinge linker to accommodate the IRE hairpin
(34). The fact that the RNA-induced protections extend in a large area
of IRP-1 further suggests that multiple contacts occur between IRP-1
and the IRE. Alternatively, protection at some residues may reflect
steric hindrance (as for Arg-149) between the proteinases and the IRE or induced conformational changes that render IRP-1 less sensitive to
proteases. Such conformational changes may explain the increased cleavages occurring in region 134-141. It is interesting to note that
this region contains a phosphorylation site at Ser-138 that occurs
preferentially in the IRE binding form of IRP-1 (25). We also showed
that IRP-1 induces some adjustments in the IRE loop promoting the
formation of a metal-ion binding site (35). Therefore the mutual
adaptation of both IRP-1 and the IRE hairpin may also contribute to the
specific recognition. Despite the similarities between IRP-1 and
mt-aconitase, the latter is not able to bind to the IRE (21) suggesting
that several crucial amino acids in the cleft are not found in the
mt-aconitase. The present data reveal that specific interactions appear
to exist between residues in the N- and C-terminal domains of IRP-1 and
the IRE. Region 720-740 contains several conserved residues among
related IRPs (Fig. 1C). Furthermore, this area of IRP-1
protein is significantly shorter in pig heart mt-aconitase, and the two
conserved arginines 721 and 728 are missing in mt-aconitase (Fig.
1C). Because arginines are frequently hydrogen-bonded with
the sugar-phosphate backbone and/or bases of RNA, they might represent
potential contact sites. From these data, one may speculate that
mutations at those specific conserved residues may alter RNA binding in
IRP-1. Conversely, their introduction in the mt-aconitase context might
confer a specific IRE binding activity to the enzyme.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-32P]ATP and T4 polynucleotide kinase (36). The RNAs
were purified on an 8% polyacrylamide, 8 M urea gel
electrophoresis, eluted overnight, and precipitated with ethanol. Prior
to use, RNAs were first incubated at 90 °C for 1 min in water and
renatured by a slow cooling at 25 °C in the appropriate buffer.
80 °C in
small aliquots.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Analysis of proteolytic digests of human
IRP-1 protein free or bound to IRE. A, autoradiogram of
a 15% protein gel showing proteolytic cleavage products on IRP-1 alone
( ) or bound to the IRE (+). Lane C denotes an incubation
control lane of IRP-1. The concentration of the proteinases used were
as follows: 0.002 unit/µl Arg-C, 0.4 ng/µl Asp-N, 0.01 µg/µl
Glu-C, 0.002 unit/µl Lys-C, or 0.02 µg/µl trypsin. More
experimental details are given under "Experimental Procedures."
Identification of corresponding amino acids are represented. To
facilitate their identification within the sequence, the amino acids
are numbered according to the N terminus of human IRP-1 and do not take
into account the His-tag residues. B, diagrams showing the
relationship between the logarithm of the mass and the migration of the
bands given in mm. Diagram B corresponds to the experiment
shown in panel A. The mass for each His-tag peptide was
calculated, and the migration was measured as the distance (in mm)
between the middle position of the band and the end of the stacking
gel. Protein markers with molecular masses of 204, 121, 78, 39.7, 30.7, 19.7, and 7.7 kDa are denoted by black dots.
C, sequence alignment for regions spanning residues 79 to
159 and 711 to 741 in related IRPs and heart pig mitochondrial
aconitase. The strictly conserved amino acids are represented by
capital letters. Proteolytic cleavages are
denoted by arrows. Numbers in parentheses are
references. The specificity of the cleavages are given in Table I.
Effect of IRE binding: O, strong protections; O, weak
protections; *, enhanced cleavages. The reduced cleavages in the
cytoplasmic aconitase form are denoted by black
spheres. Peptides 122-132 and 116-144 have been
cross-linked to IRP-1 by Basilion et al. (33) and Neupert
et al. (32), respectively.
Localization of the proteolytic cleavage sites in human IRP-1,
comparison with the aconitase form of IRP-1 (ACN), and effect of
RNA binding (RNA)
), moderate protection (
),
enhancement (+), unchanged (no symbol). The asterisks indicate the
positions of the Asp-N cleavages for which their intensities varied in
some of the experiments. The amino acids are numbered according to the
N terminus of human IRP-1 and do not take into account the His-tag
residues.
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Fig. 2.
Autoradiograms of SDS protein gels showing
the footprint obtained in the presence of the IRE or following assembly
of the iron-sulfur cluster. A, IRP-1 protein
(apoprotein) and the cytoplasmic aconitase form (holoprotein) were
digested with different proteinases (trypsin, Arg-C, Lys-C, Asp-N,
Glu-C). Reactions were done in the presence of the IRE (+) or with an
equivalent amount of tRNA ( ). Same legend as in Fig. 1. B,
IRP-1 protein (apoprotein) or the cytoplasmic aconitase form
(holoprotein) was digested with several proteinases (trypsin, Arg-C,
and Glu-C) in the presence of TRS-1 (+) or an equivalent amount of tRNA
(
). Experimental details are given under "Experimental
Procedures." C, secondary structure of the regulatory
region of TfR mRNA (TRS-G165) and of the ferritin IRE hairpin. The
minimal regulatory region of TfR mRNA is identical to the sequence
defined by Casey et al. (10) except that IRE C contains a
G165 instead of an adenine, which increased significantly the affinity
binding for IRP-1 (35). The three IREs B to D are denoted according to
Casey et al. (10). The protection induced by IRP-1 binding
are shown in the inset: protected bases are
circled and protected riboses are represented by
dots. The results are taken from Schlegl et al.
(35).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 3.
Effect of RNA binding and of iron-sulfur
cluster insertion modeled on the crystallographic structure of
mitochondrial aconitase. The coordinates are taken from Robbins
and Stout (59). Effect of RNA binding: the protected amino acids are
drawn in dark blue and yellow, and
enhanced cleavages are shown by red residues. Effect of
iron-sulfur insertion: the reduced cleavages as compared with IRP-1 are
shown by yellow residues. The residues were assigned based
on sequence comparison between human IRP-1 and mitochondrial aconitase
(23, 27). Residues 122 to 132 shown in light blue
have been cross-linked to the ferritin IRE (32, 33).
The footprinting approach also provides further evidence for significant structural changes of IRP-1 induced by the iron-sulfur cluster. As previously shown, the reconstitution of the iron-sulfur cluster in IRP-1 precludes RNA binding and confers aconitase activity. In good agreement with the study of Schalinske et al. (25), the c-aconitase form is more resistant to the action of proteases. It is of interest that the strongest reductions of cleavages were located in the two regions found protected by the IRE, mainly at Asp-125, Arg-149, Glu-155, and Arg-187 in the N-terminal domain and at Arg-721, Arg-728, Arg-732, and Asp-751 in the C-terminal domain. Because these residues are located far away from the three cysteines (437, 503, 506) that have been shown essential for catalysis (29, 30, 51), the protection may reflect conformational changes resulting in protein structure that is less sensitive to proteinases. All these residues lie in the putative cleft of IRP-1 (Fig. 3) and are also located close to the two phosphorylation sites (25). These data indicate that the enzymatic form of IRP-1 adopts a closed conformation stabilized by the presence of the iron-sulfur cluster. This is consistent with the crystal structure of mt-aconitase, which showed the presence of a narrow cleft stabilized by interactions involving side chain residues of domain 4 and domains 1-3 (28). Conversely, in the absence of the iron-sulfur cluster, residues located in the cleft (in the N-terminal domain) and at the edge of the cleft (in domain 4) become accessible to the solvent, permitting the access of the IRE.
In summary, our data suggest the presence of a bipartite IRE binding
site in human IRP-1 and illustrate the potential of the protein to
alter its conformation in its different functional states. Obviously,
the exact nature of these conformational changes and of the IRE-IRP-1
contact sites will require the crystallographic structure of IRE-IRP-1 complex.
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ACKNOWLEDGEMENTS |
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We are grateful to Steve Lodmell and Jean-Marc Lanchy for helpful discussions and critical reading of the manuscript. We also thank Christine Lichte for protein sequencing and Philippe Walter for the help in protein sequence alignment.
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FOOTNOTES |
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* This work was supported by grants from the Association pour la Recherche sur le Cancer under ARC contrat 9346.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
These two authors contributed equally to this work.
§ Supported by a long term fellowship from the European Molecular Biology Organization.
To whom correspondence and reprint requests should be
addressed. E-mail: romby{at}ibmc.u-strasbg.fr.
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ABBREVIATIONS |
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The abbreviations used are: IRP, iron regulatory protein; IRE, iron-responsive element; TfR, transferrin receptor; Tricine, N-tris(hydroxymethyl)methylglycine; PAGE, polyacrylamide gel electrophoresis; mt, mitochondrial; c, cytoplasmic.
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REFERENCES |
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