Alternative Structural State of Transferrin
THE CRYSTALLOGRAPHIC ANALYSIS OF IRON-LOADED BUT DOMAIN-OPENED OVOTRANSFERRIN N-LOBE*

Kimihiko Mizutani, Honami Yamashita, Hirofumi KurokawaDagger , Bunzo Mikami, and Masaaki Hirose§

From the Research Institute for Food Science, Kyoto University, Uji, Kyoto 6110011, Japan

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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-. 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
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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- (2-10).

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-, 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
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 CuKalpha 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).

                              
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Table I
Summary of data collection and refinement

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 - 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).

The omit maps (2Fo - Fc, contoured at 1 sigma  and Fo - Fc, contoured at 3 sigma ) 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 sigma  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
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 - Fc electron density map, there is no break in the main chain density when contoured at the 1 sigma  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 sigma  (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.

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 (phi  = 75.0°, psi  = -52.2°). This leucine residue is the central residue in a gamma -turn. The gamma -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 gamma -turn residue, Leu299, is labeled.

Overall Organization of the Structure-- Fig. 2 displays the overall structure of ovotransferrin N-lobe as a Calpha 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 Calpha 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 beta -strands linking the domains.


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Fig. 2.   Stereo Calpha 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.

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 - 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 sigma ; blue: Fo - Fc, contoured at 3 sigma ) 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 sigma  (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.

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).


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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.

                              
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Table II
Geometry of the iron binding site

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- 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
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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).
<UP>apo-Trf</UP>+<UP>Fe<SUP>3+</SUP></UP> · <UP>NTA ⇌ Trf</UP> · <UP>Fe<SUP>3+</SUP></UP> · <UP>NTA</UP> (Reaction 1)
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-; this reaction yields the physiological holo form consisting of transferrin, Fe3+, and CO32- (Trf·Fe3+·CO32-) (1, 28).
   <UP>Trf</UP> · <UP>Fe<SUP>3+</SUP></UP> · <UP>NTA</UP>+<UP>CO</UP><SUP><UP>2−</UP></SUP><SUB><UP>3</UP></SUB><UP> ⇌ Trf</UP> · <UP>Fe<SUP>3+</SUP></UP> · <UP>CO</UP><SUP><UP>2−</UP></SUP><SUB><UP>3</UP></SUB>+<UP>NTA</UP> (Reaction 2)
The current crystal structure of the Fe3+-soaked form demonstrates essentially the same open conformation as apo-Trf, whereas Trf·Fe3+·CO32- 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.

    ACKNOWLEDGEMENT

Computational time was provided by the Super-Computer Laboratory, Institute for Chemical Research, Kyoto University.

    FOOTNOTES

* 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.

Dagger 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.

    ABBREVIATIONS

The abbreviations used are: NTA, nitrilotriacetate; BisTris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)-propane-1,3-diol; Trf, transferrin.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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