Alternative Structural State of Transferrin

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 CO3 2−. 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.

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 Fe 3ϩ ions (1). They are ϳ80-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)(3)(4)(5)(6) and the monoferric N-lobes (7-10) of several transferrins reveal that the two domains are closed over an Fe 3ϩ ion. Four of the six Fe 3ϩ coordination sites are occupied by the protein ligands of two tyrosine residues, one aspartic acid residue, and one histidine residue (Asp 60 , Tyr 92 , Tyr 191 , and His 250 in ovotransferrin N-lobe) and the other two, by a synergistic anion, physiologically bidentate CO 3 2Ϫ (2-10). For the iron-free apo form, however, x-ray crystallographic (11)(12)(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 Fe 3ϩ 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 (Asp 60 in the domain 1,Tyr 191 in the domain 2, and Tyr 92 and His 250 in different hinges) (2)(3)(4)(5)(6)(7)(8)(9)(10) inevitably require an alternative Fe 3ϩ coordination structure for the Fe 3ϩ -loaded, domain-opened intermediate. Such an alternative structural state has been a central question to be solved for the understanding of the Fe 3ϩ 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 Fe 3ϩ coordination. An Fe 3ϩ -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 hololike closed conformation (20,21). The mutant lactoferrin N-lobe in which the synergistic anion-binding residue, Arg 121 , 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 Fe 3ϩ soaking conditions in which the colorless crystal turns red without any collapse were searched. As a successful condition, an apo crystal of ovotransferrin Nlobe was soaked with the Fe 3ϩ ⅐NTA 1 complex in the absence of CO 3 2Ϫ , and then its structure was solved at a 2.1 Å resolution. We report here a novel structural state of transferrin: the Fe 3ϩ -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 Tyr 92 -OH and Tyr 191 -OH. Other Fe 3ϩ 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 Fe 3ϩbinding ligands in the open transferrin.

EXPERIMENTAL PROCEDURES
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 l of protein solution (44.4 mg/ml in 0.05 M BisTris-HCl buffer, pH 6.0) with 5 l 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 Fe 3ϩ 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 Fe 3ϩ ⅐NTA for 4 h, thereby being transformed into red ones.
Data Collection and Processing-Diffraction data were collected using CuK ␣ radiation (ϭ 1.5418 Å) with a Siemens Hi-Star area detector coupled to a rotating anode generator (Mac Science M18XHF). The crystal of the Fe 3ϩ -soaked form was found to belong to the same space group of P6 3 22 (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).
Model Building and Refinement-As the model structure, we employed the apo structure of ovotransferrin N-lobe 2 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 Fe 3ϩ -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 Ϫ 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 Ϫ Fc, contoured at 1 and Fo Ϫ Fc, contoured at 3 ) were obtained using the reflection data of the Fe 3ϩ -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 Fe 3ϩsoaked 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.

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 Ϫ 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.
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, Leu 299 lies outside the allowed regions ( ϭ 75.0°, ϭ Ϫ52.2°). 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).
Overall Organization of the Structure- Fig. 2 displays the overall structure of ovotransferrin N-lobe as a C ␣ trace. The overall structure of the Fe 3ϩ -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 Fe 3ϩ -and CO 3 2Ϫ -loaded form) structure of ovotransferrin N-lobe (8), comprise a domainopened 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 hen 3 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.
Another important observation in Fig. 2 is that an iron atom exists in the opened interdomain cleft of the Fe 3ϩ -soaked form. The two Fe 3ϩ -ligating tyrosine residues in the holo form (Tyr 92 and Tyr 191 ) appear also to participate in the iron coordination in the Fe 3ϩ -soaked form, whereas the other two protein ligands of Asp 60 and His 250 are located quite far from the iron atom.
The Structure of the Fe 3ϩ Binding Site-The Fe 3ϩ binding structure was investigated in more details for the Fe 3ϩ -soaked form. Fig. 3a is a stereo  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 Fe 3ϩ -soaked form. As summarized in Table II, the distances from the iron of Tyr 92 -OH and Tyr 191 -OH are 1.90 Å and 1.76 Å, respectively, indicating the Fe 3ϩ coordination by these two tyrosine residues. The other

FIG. 2. Stereo C ␣ plots of apo (black) and Fe 3؉ -soaked (cyan) and holo forms (red) of ovotransferrin N-lobe.
The figures are produced with MOL-SCRIPT (30) and Raster3D (31) as the superimposed ones on domain N2. The holo (Fe 3ϩ -and CO 3 2Ϫ -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 Fe 3ϩ -soaked form (see "Experimental Procedures"). The residue numbers are labeled for the Fe 3ϩsoaked form. The iron atom (green sphere) and the side chains (blue) of His 250 , Asp 60 , Tyr 92 , and Tyr 191 (from top to bottom in this order) for the Fe 3ϩ -soaked form are also displayed.  (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 Ala 123 -N, and NTA-O13, to Thr 117 -OG1 and Gly 124 -N. These protein groups are the ones that form the hydrogen bonds with CO 3 2Ϫ anion in the holo form (Fig. 4b). As a surprising observation, however, NTA has no direct interaction with the synergistic anion-binding residue, Arg 121 . Another difference in the protein-anion interactions is that the hydrogen bond of NTA-O4 to Ser 122 -N in the Fe 3ϩ -soaked form is replaced by that of Asp 60 -OD2 to Ser 122 -N in the holo form.
The Trf⅐Fe 3ϩ ⅐NTA 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 CO 3 2Ϫ ; this reaction yields the physiological holo form consisting of transferrin, Fe 3ϩ , and CO 3 2Ϫ (Trf⅐Fe 3ϩ ⅐CO 3 2Ϫ ) (1, 28).
The current crystal structure of the Fe 3ϩ -soaked form demonstrates essentially the same open conformation as apo-Trf, whereas Trf⅐Fe 3ϩ ⅐CO 3 2Ϫ assumes the closed one (Fig. 2). About the implications of the Fe 3ϩ -soaked structure for the iron binding pathway, two different mechanisms may be possible.
In the first mechanism, Trf⅐Fe 3ϩ ⅐NTA assumes the same conformation in solution as the Fe 3ϩ -soaked structure, and the total domain closure occurs in Reaction 2. In this mechanism, the domain closure should depend on the anion replacement. The Trf⅐Fe 3ϩ ⅐NTA complex shares the two protein ligands (Tyr 92 and Tyr 191 residues) with Trf⅐Fe 3ϩ ⅐CO 3 2Ϫ (Fig. 4). Nev- FIG. 4. Diagram of the Fe 3؉ coordination and hydrogen bonding network. a, the Fe 3ϩ -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 Fe 3ϩ , 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   ertheless, 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, CO 3 2Ϫ forms hydrogen bonds with Thr 117 -OG1, Ala 123 -N, and Gly 124 -N in the holo form; these protein groups are all hydrogen bonded to carboxylate groups of NTA in the Fe 3ϩ -soaked form (Fig. 4a). The protein group Ser 122 -N that forms a hydrogen bond with Asp 60 -OD2 in the holo form also forms a hydrogen bond with NTA-O4 in the Fe 3ϩ -soaked form. However, Arg 121 -NE and -NH 2 , which are the anchor sites for CO 3 2Ϫ in the holo form, are both vacant in the Fe 3ϩsoaked form. Such an open situation would be suitable for the subsequent entry of CO 3 2Ϫ in Reaction 2. Our putative pathway for Reaction 2 includes an initial entry of CO 3 2Ϫ into the Arg 121 anchor sites and then the total replacement of NTA by CO 3 2Ϫ . This reaction should yield a short lived Trf⅐Fe 3ϩ ⅐CO 3 2Ϫ complex with the open conformation, in which only four of the six Fe 3ϩ coordination sites are occupied by the protein side chains (Tyr 92 and Tyr 191 ) and bidentate CO 3 2Ϫ . 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 Fe 3ϩ coordination structure by the two tyrosine residues and CO 3 2Ϫ (29). The formation of the holo structure is then completed by the coordination of Asp 60 and His 250 ligands to the two vacant Fe 3ϩ sites and hence by the domain closure.
In the second mechanism, the Fe 3ϩ -soaked form is initially transformed into a closed conformation by the Fe 3ϩ coordination of Asp 60 and His 250 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 Trf⅐Fe 3ϩ ⅐NTA complex with the closed conformation in a carbonate-free condition; the maintenance of the open conformation in the Fe 3ϩ -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 Trf⅐Fe 3ϩ ⅐NTA complex because the Asp 60 and His 250 coordination sites are occupied by NTA in the Fe 3ϩ -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 Fe 3ϩ ⅐NTA 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 Trf⅐Fe 3ϩ ⅐CO 3 2Ϫ . 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 Trf⅐Fe 3ϩ ⅐NTA complex with a hololike closed conformation in solution cannot be ruled out because the structure of the Trf⅐Fe 3ϩ ⅐NTA 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, Fe 3ϩ -loaded holo form (2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13). As an alternative structural state, the current Fe 3ϩ -soaked structure of the ovotransferrin N-lobe is the first demonstration of an Fe 3ϩ -loaded, open transferrin form. One of the most important findings in the current structure is that only the two tyrosine residues (Try 92 and Tyr 191 ) participate in the Fe 3ϩ 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 Fe 3ϩloaded, 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 Trf⅐Fe 3ϩ ⅐NTA complex assumes the open or closed conformation in solution, the finding that the overall structure of the Fe 3ϩ -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 Fe 3ϩ entry in the intact transferrin lobe with the domain-opened conformation.