From the Research Institute for Food Science, Kyoto University, Uji, Kyoto 6110011, Japan
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ABSTRACT |
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Transferrins bind Fe3+ very
tightly in a closed interdomain cleft by the coordination of four
protein ligands (Asp60, Tyr92,
Tyr191, and His250 in ovotransferrin N-lobe)
and of a synergistic anion, physiologically bidentate
CO32 Transferrins are a group of iron-binding proteins which includes
serum transferrin, lactoferrin, and ovotransferrin (1). The proteins
serve to control the iron level in the body fluid of vertebrates by
their ability to bind very tightly two Fe3+ ions (1). They
are ~80-kDa single-chain proteins and consist of two similarly sized
homologous N- and C-lobes, which are further divided into two similarly
sized domains (domains N1 and N2 in the N-lobe and domains C1 and C2 in
the C-lobe). The two iron binding sites are located within the
interdomain cleft of each lobe. Crystal structures of the diferric
forms (2-6) and the monoferric N-lobes (7-10) of several transferrins
reveal that the two domains are closed over an Fe3+ ion.
Four of the six Fe3+ coordination sites are occupied by the
protein ligands of two tyrosine residues, one aspartic acid residue,
and one histidine residue (Asp60, Tyr92,
Tyr191, and His250 in ovotransferrin N-lobe)
and the other two, by a synergistic anion, physiologically bidentate
CO32 For the iron-free apo form, however, x-ray crystallographic (11-13)
and solution scattering (14-17) analyses have revealed that all of the
transferrin lobes, except for the lactoferrin C-lobe in crystal, assume
a conformation with an opening of the interdomain cleft. This implies
that transferrin initially binds the Fe3+ ion in the open
form before being transformed into the closed holo form (18, 19).
Differential domain and hinge locations of the four protein ligands
(Asp60 in the domain 1, Tyr191 in the domain 2, and Tyr92 and His250 in different hinges)
(2-10) inevitably require an alternative Fe3+ coordination
structure for the Fe3+-loaded, domain-opened intermediate.
Such an alternative structural state has been a central question to be
solved for the understanding of the Fe3+ binding pathway in transferrin.
A major difficulty encountered in the structural analysis for the
intermediate is to prepare a stable protein form that reasonably mimics
it. One of the most promising ways may be the site-directed mutagenesis
approach for the amino acid residues that are implicated in the
Fe3+ coordination. An Fe3+-loaded,
domain-opened transferrin form, however, has not been obtained so far
by site-directed mutagenesis; either the Asp- or His-ligand mutant of
the lactoferrin N-lobe assumes the holo-like closed conformation (20,
21). The mutant lactoferrin N-lobe in which the synergistic
anion-binding residue, Arg121, is replaced by the serine or
glutamic acid residue also assumes the closed conformation (22).
In the present study, we employed an alternative strategy using an apo
crystal: the Fe3+ soaking conditions in which the colorless
crystal turns red without any collapse were searched. As a successful
condition, an apo crystal of ovotransferrin N-lobe was soaked with the
Fe3+·NTA1
complex in the absence of CO32 Crystallization--
The isolated N-lobe (N-terminal
half-molecule) of hen ovotransferrin was purified as described (23).
The apo form of the protein was crystallized using the hanging drop
vapor diffusion method. A solution of a crystallization droplet was
prepared on a siliconized coverslip by mixing 5 µl of protein
solution (44.4 mg/ml in 0.05 M BisTris-HCl buffer, pH 6.0)
with 5 µl of precipitant solution (0.05 M BisTris buffer,
pH 6.0, 52% ammonium sulfate). The droplets were equilibrated against
1 ml of the precipitant solution at 20 °C. Hexagonal apo crystals
were obtained within 1 month. For Fe3+ soaking, the apo
crystals were first transferred to a precipitant solution at higher pH
(0.05 M BisTris-HCl buffer, pH 7.5, 52% ammonium sulfate)
and then stepwise to raised precipitant concentrations (56, 62, 68, 74, and then 80% ammonium sulfate). The colorless crystals were incubated
at 20 °C with 3.0 mM Fe3+·NTA for 4 h, thereby being transformed into red ones.
Data Collection and Processing--
Diffraction data were
collected using CuK Model Building and Refinement--
As the model structure, we
employed the apo structure of ovotransferrin
N-lobe2 that had been solved
at 1.9 Å resolution by the isomorphous replacement method using the
hexagonal apo crystals. Using the apo structure model and the
diffraction data of the Fe3+-soaked form, refinement
calculations were carried out by X-PLOR (24). One NTA molecule and one
iron atom, which were identified from a clear difference density
(Fo
The omit maps (2Fo Quality of the Final Model--
The N-lobe of ovotransferrin
comprises 332 amino acid residues (23). Residues 1-3, however, are not
included in the final model because no clearly interpretable electron
density could be seen for these residues. In the final
2Fo
A Ramachandran plot (25) of the main chain torsion angles is shown in
Fig. 1; 88.3% of the residues are in the
core regions, with 99.3% of the residues lying within the allowed
regions as defined in the program PROCHECK (26). As a non-glycine
residue, Leu299 lies outside the allowed regions ( Overall Organization of the Structure--
Fig.
2 displays the overall structure of
ovotransferrin N-lobe as a C
Another important observation in Fig. 2 is that an iron atom exists in
the opened interdomain cleft of the Fe3+-soaked form. The
two Fe3+-ligating tyrosine residues in the holo form
(Tyr92 and Tyr191) appear also to participate
in the iron coordination in the Fe3+-soaked form, whereas
the other two protein ligands of Asp60 and
His250 are located quite far from the iron atom.
The Structure of the Fe3+ Binding Site--
The
Fe3+ binding structure was investigated in more details for
the Fe3+-soaked form. Fig.
3a is a stereo diagram of the
electron density map calculated with the exclusion of the NTA model
(green, 2Fo
To evaluate the existence of an iron atom by an alternative way, we
calculated the anomalous difference Fourier density map with exclusion
of an iron atom. As shown in Fig. 3b (purple), the existence of iron atom is clearly confirmed by the highest anomalous difference Fourier peak in the density map.
Fig. 4a is a diagram
displaying the iron coordination and hydrogen bonding structure in the
Fe3+-soaked form. As summarized in Table
II, the distances from the iron of
Tyr92-OH and Tyr191-OH are 1.90 Å and 1.76 Å,
respectively, indicating the Fe3+ coordination by these two
tyrosine residues. The other two protein ligands of Asp60
and His250, however, are not involved in the coordination
of the iron atom: the distances of Asp60-OD1 and
His250-NE2 from iron are 9.09 Å and 8.47 Å, respectively.
Instead, at least three of the other four Fe3+ coordination
sites are occupied by NTA; the distances from iron of NTA-O5, NTA-O8,
and NTA-O12 are 2.03 Å, 1.73 Å, and 2.22 Å, respectively (Table II).
The last coordination site may be weakly coordinated by NTA-N1 or
almost vacant because the distance of this ligand from iron is a
slightly larger value of 2.76 Å, compared with the other coordination
distances (Table II). The bond angles formed among NTA-N1, iron, and
NTA-O (O5, O8, or O12) are also significantly apart from the ideal
90° (Table II).
The binding of NTA is stabilized through the interactions with the
protein chains: NTA-O12 is hydrogen bonded to Ala123-N, and
NTA-O13, to Thr117-OG1 and Gly124-N. These
protein groups are the ones that form the hydrogen bonds with
CO32 Previous x-ray crystallographic (2-13) and solution scattering
(14-17) analyses have revealed that upon Fe3+ uptake,
transferrins undergo a large scale conformational transition from the
domain-opened apo structure into the closed holo structure. The
structural pathway for the large conformational transition, however,
has not been known. The current Fe3+-soaked structure
provides crucial information about the structural mechanism for the
Fe3+ binding.
For loading Fe3+ to apotransferrin (apo-Trf), a ferric
chelate, most widely ferric NTA (Fe3+·NTA), is employed
(1). The binding reaction yields a ternary complex consisting of
transferrin, Fe3+, and NTA molecules
(Trf·Fe3+·NTA) (1, 28).
. Upon Fe3+
uptake, transferrins undergo a large scale conformational transition: the apo structure with an opening of the interdomain cleft is transformed into the closed holo structure, implying initial
Fe3+ binding in the open form. To solve the
Fe3+-loaded, domain-opened structure, an ovotransferrin
N-lobe crystal that had been grown as the apo form was soaked with
Fe3+-nitrilotriacetate, and its structure was solved at 2.1 Å resolution. The Fe3+-soaked form showed almost exactly
the same overall open structure as the iron-free apo form. The electron
density map unequivocally proved the presence of an iron atom with the
coordination by the two protein ligands of Tyr92-OH and
Tyr191-OH. Other Fe3+ coordination sites are
occupied by a nitrilotriacetate anion, which is stabilized through the
hydrogen bonds with the peptide NH groups of Ser122,
Ala123, and Gly124 and a side chain group of
Thr117. There is, however, no clear interaction between the
nitrilotriacetate anion and the synergistic anion binding site,
Arg121.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(2-10).
, and
then its structure was solved at a 2.1 Å resolution. We report here a
novel structural state of transferrin: the Fe3+-loaded
structure of ovotransferrin N-lobe with essentially the same open
conformation as the apo form. In this structure, the bound iron atom is
coordinated by the two protein ligands of Tyr92-OH and
Tyr191-OH. Other Fe3+ coordination sites are
occupied by a NTA anion, which is stabilized through the hydrogen bonds
with protein groups. The observation strongly suggests that the two
tyrosine residues are the initial Fe3+-binding ligands in
the open transferrin.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
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radiation (= 1.5418 Å) with a
Siemens Hi-Star area detector coupled to a rotating anode generator
(Mac Science M18XHF). The crystal of the Fe3+-soaked form
was found to belong to the same space group of P6322 (Table
I) as that of the apo form. 188,024 reflections were collected to 2.09 Å. The data were processed,
merged, and scaled with the SAINT program (Siemens Analytical x-ray
Instruments, Inc., Madison, WI).
Summary of data collection and refinement
Fc) map of the first refinement round, were included in the model, followed by more than 10 rounds of
refinements and manual model buildings. The parameter and topology files of NTA for X-PLOR (24) were prepared after building and energy
minimization of NTA by QUANTA and CHARM (Molecular Simulations Inc.,
San Diego, CA).
Fc, contoured at 1
and Fo
Fc, contoured at 3
) were
obtained using the reflection data of the Fe3+-soaked form
at 7.0-2.1 Å resolution after refinement of the model in which the
NTA molecule was excluded. An anomalous difference Fourier density map
contoured at 3
was calculated with a separate data set of the
Fe3+-soaked form at 7.0-2.1 Å resolution with pair
completeness of 95.8% by the program PHASES. The phases were
calculated from the final model without an iron atom by X-PLOR and
merged with the data by PHASES.
RESULTS
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ABSTRACT
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EXPERIMENTAL PROCEDURES
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Fc electron density map, there is no
break in the main chain density when contoured at the 1
level.
Relevant refinement statistics are given in Table I. The overall
completeness, R factor, and free-R value were
88.3%, 0.189, and 0.256, respectively, for the data more than 2
(F). For the highest resolution bin (2.10-2.19 Å), the completeness was 75.5%, and the R factor and
free-R value were, respectively, 0.262 and 0.274. From a
Luzzati plot, the mean absolute error in atomic position is estimated
to be 0.24 Å.
= 75.0°,
=
52.2°). This leucine residue is the central residue
in a
-turn. The
-turn of the equivalent leucine residue is the
one conserved in all of the N- and C-lobes of serum transferrin (2),
ovotransferrin (4), and lactoferrin (6).
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Fig. 1.
Ramachandran plot of the backbone torsion
angles. Glycine residues are represented by triangles
and non-glycine residues as squares. The -turn residue,
Leu299, is labeled.
trace. The overall
structure of the Fe3+-soaked form was almost exactly the
same as that of the apo form. The root mean square
deviation for 329 C
atoms was only 0.19 Å. These
structures, when compared with the holo (the Fe3+- and
CO32
-loaded form) structure of
ovotransferrin N-lobe (8), comprise a domain-opened conformation (Fig.
2). The extent and mode of the opening were almost the same as in the
N-lobes of the whole molecules of lactoferrin (11) and duck (12) and
hen3 ovotransferrin: as
calculated by the rigid body motion method (27), the domains move
49.7° around a rotation axis passing through the two
-strands
linking the domains.
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Fig. 2.
Stereo C
plots of apo (black) and Fe3+-soaked
(cyan) and holo forms (red) of
ovotransferrin N-lobe. The figures are produced with MOLSCRIPT
(30) and Raster3D (31) as the superimposed ones on domain N2. The holo
(Fe3+- and CO32
-loaded
ovotransferrin N-lobe) structure is drawn using the previous data (8).
The apo structure of ovotransferrin N-lobe is the one employed as the
model for the current structural determination of the
Fe3+-soaked form (see "Experimental Procedures"). The
residue numbers are labeled for the Fe3+-soaked form. The
iron atom (green sphere) and the side chains
(blue) of His250, Asp60,
Tyr92, and Tyr191 (from top to
bottom in this order) for the Fe3+-soaked form
are also displayed.
Fc;
blue, Fo
Fc) for the
Fe3+-soaked form. The figure clearly demonstrates the
existence of iron and NTA close to the Tyr92 and
Tyr191 ligands.
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Fig. 3.
Stereo views depicting the iron binding site
in the Fe3+-soaked form. a, electron
density maps (green: 2Fo Fc,
contoured at 1
; blue: Fo
Fc, contoured at 3
) obtained using the reflection data
of the Fe3+-soaked form after refinement of the model in
which the NTA molecule was omitted. b, anomalous difference
Fourier density map contoured at 3
(purple) calculated
with exclusion of an iron atom using the reflection data of the
Fe3+-soaked form at 7.0-2.1 Å. The final model is
superimposed in stick presentation with atoms in standard colors.
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Fig. 4.
Diagram of the Fe3+ coordination
and hydrogen bonding network. a, the
Fe3+-soaked form; b, the holo form. The diagram
for the holo form is drawn using the previous data (4) for comparison.
The thick solid lines in black represent the
possible coordination to Fe3+, although the coordination by
NTA-N is not clear because of a longer iron to ligand distance (2.76 Å) than the other coordination distances (see Table II). The
thin solid lines in black display hydrogen bonds
with the bond distances in Å. The synergistic anions (NTA in
a and CO32 in b)
are shown in red. The protein chains are shown in
blue. The numbers 60 and 250 in
b represent Asp60 and His250
ligands, respectively.
Geometry of the iron binding site
anion in the holo form (Fig.
4b). As a surprising observation, however, NTA has no direct
interaction with the synergistic anion-binding residue,
Arg121. Another difference in the protein-anion
interactions is that the hydrogen bond of NTA-O4 to
Ser122-N in the Fe3+-soaked form is replaced by
that of Asp60-OD2 to Ser122-N in the holo form.
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
The Trf·Fe3+·NTA complex is a stable form in the
absence of other synergistic anions. In the presence of a high
concentration of bicarbonate, however, NTA is replaced by
CO32
(Reaction 1)
; this reaction yields the
physiological holo form consisting of transferrin, Fe3+,
and CO32
(Trf·Fe3+·CO32
) (1,
28).
The current crystal structure of the Fe3+-soaked form
demonstrates essentially the same open conformation as apo-Trf, whereas Trf·Fe3+·CO32
(Reaction 2)
assumes
the closed one (Fig. 2). About the implications of the Fe3+-soaked structure for the iron binding pathway, two
different mechanisms may be possible.
In the first mechanism, Trf·Fe3+·NTA assumes the same
conformation in solution as the Fe3+-soaked structure, and
the total domain closure occurs in Reaction 2. In this mechanism, the
domain closure should depend on the anion replacement. The
Trf·Fe3+·NTA complex shares the two protein ligands
(Tyr92 and Tyr191 residues) with
Trf·Fe3+·CO32 (Fig.
4). Nevertheless, some structural modulations in the iron binding site,
other than the protein ligand structures, appear to be highly relevant
to the structural mechanism in Reaction 2. As displayed in Fig.
4b, CO32
forms hydrogen
bonds with Thr117-OG1, Ala123-N, and
Gly124-N in the holo form; these protein groups are all
hydrogen bonded to carboxylate groups of NTA in the
Fe3+-soaked form (Fig. 4a). The protein group
Ser122-N that forms a hydrogen bond with
Asp60-OD2 in the holo form also forms a hydrogen bond with
NTA-O4 in the Fe3+-soaked form. However,
Arg121-NE and -NH2, which are the anchor sites
for CO32
in the holo form, are both
vacant in the Fe3+-soaked form. Such an open situation
would be suitable for the subsequent entry of
CO32
in Reaction 2. Our putative
pathway for Reaction 2 includes an initial entry of
CO32
into the Arg121
anchor sites and then the total replacement of NTA by
CO32
. This reaction should yield a
short lived
Trf·Fe3+·CO32
complex
with the open conformation, in which only four of the six
Fe3+ coordination sites are occupied by the protein side
chains (Tyr92 and Tyr191) and bidentate
CO32
. As a structural counterpart, the
crystal structure of a domain 2 fragment complex, in which all parts of
domain 1 as well as the aspartic acid and histidine ligands are deleted
by proteolysis, demonstrates the presence of an equivalent
Fe3+ coordination structure by the two tyrosine residues
and CO32
(29). The formation of the
holo structure is then completed by the coordination of
Asp60 and His250 ligands to the two vacant
Fe3+ sites and hence by the domain closure.
In the second mechanism, the Fe3+-soaked form is initially
transformed into a closed conformation by the Fe3+
coordination of Asp60 and His250 ligands,
before the bound NTA molecule is replaced by a carbonate anion. This
mechanism, however, requires as a prerequisite the occurrence, in
solution, of the differential ternary Trf·Fe3+·NTA
complex with the closed conformation in a carbonate-free condition; the
maintenance of the open conformation in the Fe3+-soaked
form is accounted for by the arrest due to a crystal packing force from
otherwise (free in solution) induced transformation into the closed
conformation. The mechanism would also require a rearranged
coordination and hydrogen bonding structure for NTA in the
Trf·Fe3+·NTA complex because the Asp60 and
His250 coordination sites are occupied by NTA in the
Fe3+-soaked form (Figs. 3 and 4). As a related observation
to the second mechanism, no clear diffraction has been detected for the apo crystal when it is soaked with Fe3+·NTA in the
presence of a high concentration of bicarbonate. This strongly suggests
that the apo crystal collapses upon the transformation of the open form
into the closed
Trf·Fe3+·CO32. The
crystal packing force, therefore, may not be strong enough to arrest
the transformation of the open to closed conformation, giving less
weight to the second mechanism. At the present stage, however, the
second mechanism, which includes a ternary Trf·Fe3+·NTA
complex with a holo-like closed conformation in solution cannot be
ruled out because the structure of the Trf·Fe3+·NTA
complex in solution has not been solved.
In conclusion, the previous crystallographic structures of transferrins
have been restricted essentially to the two structural forms: one is
the domain-opened, iron-free apo form and the other, the domain-closed,
Fe3+-loaded holo form (2-13). As an alternative structural
state, the current Fe3+-soaked structure of the
ovotransferrin N-lobe is the first demonstration of an
Fe3+-loaded, open transferrin form. One of the most
important findings in the current structure is that only the two
tyrosine residues (Try92 and Tyr191)
participate in the Fe3+ coordination as protein ligands
(Figs. 3 and 4 and Table II). The coordination structure by the two
tyrosine residues is consistent with the previous hypothetical
structure for the Fe3+-loaded, domain-opened transferrin
intermediate (19); the hypothetical structure has been derived from the
crystallographic data of the iron-loaded domain N2 fragment of duck
ovotransferrin in which the Asp and His ligands, but not the two Tyr
ligands, are removed by proteolysis (29). Regardless of whether the
Trf·Fe3+·NTA complex assumes the open or closed
conformation in solution, the finding that the overall structure of the
Fe3+-soaked form is almost indistinguishable from that of
the apo form (Fig. 2) is consistent with the view that the two
tyrosine residues are the protein ligands for the
Fe3+ entry in the intact transferrin lobe with the
domain-opened conformation.
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ACKNOWLEDGEMENT |
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Computational time was provided by the Super-Computer Laboratory, Institute for Chemical Research, Kyoto University.
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FOOTNOTES |
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* This work was supported in part by a grant-in-aid for scientific research from the Ministry of Education, Science, Sports, and Culture of Japan.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.
The atomic coordinates of the apo and Fe3+-soaked forms (codes 1TFA and 1NFT) have been deposited in the Protein Data Bank, Brookhaven National Laboratory, Upton, NY.
Present address: Division of Molecular and Structural Biology,
Ontario Cancer Institute and Department of Medical Biophysics, University of Toronto, 610 University Ave., Toronto, Ontario, Canada
M5G 2M9.
§ To whom correspondence should be addressed. Tel.: 81-774-38-3734; Fax: 81-774-38-3735; E-mail: hirose{at}soya.food.kyoto-u.ac.jp.
2 K. Mizutani, H. Yamashita, B. Mikami, and M. Hirose, manuscript in preparation.
3 H. Kurokawa, J. C. Dewan, B. Mikami, J. C. Sacchettini, and M. Hirose, manuscript in preparation.
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ABBREVIATIONS |
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The abbreviations used are: NTA, nitrilotriacetate; BisTris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)-propane-1,3-diol; Trf, transferrin.
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