Ti(IV) uptake and release by human serum transferrin and recognition of Ti(IV)-transferrin by cancer cells: understanding the mechanism of action of the anticancer drug titanocene dichloride

The organometallic anticancer agent titanocene dichloride, Cp(2)TiCl(2), is now in phase II clinical trials as an anticancer drug, but its mechanism of action is poorly understood. We show here that the interactions of Cp(2)TiCl(2) with human serum transferrin (hTF) and that of Ti(2)-hTF with adenosine triphosphate (ATP) have characteristics that could allow transferrin to act as a mediator for titanium delivery to tumor cells. Such reactions may therefore be important to the anticancer activity of this new class of drugs. Cp(2)TiCl(2) reacts rapidly with human apo-transferrin under physiological conditions (100 mM NaCl, 25 mM bicarbonate, and 4 mM phosphate, pH 7.4) with carbonate as a synergistic anion. The Cp ligands are released from the drug. Two-dimensional [(1)H, (13)C] NMR studies of epsilon-[(13)C]Met-hTF show that Ti(IV) loads the C-lobe first followed by the N-lobe and binds in the specific Fe(III) sites. The protein conformational changes induced by Ti(IV) appear to be similar to those induced by Fe(III). Carbonate can act as a synergistic anion in Ti(2)-hTF but does not appear to be essential. A specific Ti(IV)-hTF adduct is formed even in the absence of bicarbonate. When the pH of Ti(2)-hTF solutions is lowered, no Ti(IV) is released at the endosomal pH of ca. 5.0-5.5, but one Ti(IV) dissociates between pH 4.5-2.0. In contrast, in the presence of 1 mM ATP, all Ti(IV) is readily released from both lobes when the pH is lowered from 7.0 to 4.5. Moreover, Fe(III) displaces Ti(IV) rapidly from the C-lobe of Ti(2)-hTF (<5 min) but only slowly (days) from the N-lobe. Thus, the species Fe(C)Ti(N)-hTF might also provide a route for Ti(IV) entry into tumor cells via the transferrin receptor. Ti(2)-hTF effectively blocked cell uptake of radiolabeled (59)Fe-hTF into BeWo cells, a human placental choriocarcinoma cell line in culture. These results imply that titanium transferrin might be recognized by the transferrin receptor and be taken up into cancer cells.     

X-ray crystallography and mass spectroscopy reveal that the N-lobe of human transferrin expressed in Pichia pastoris is folded correctly but is glycosylated on serine-32

The ferric form of the N-lobe of human serum transferrin (Fe(III)-hTF/2N) has been expressed at high levels in Pichia pastoris. The Fe(III)-hTF/2N was crystallized in the space group P41212, and X-ray crystallography was used to solve the structure of the recombinant protein at 2.5 A resolution. This represents only the second P. pastoris-derived protein structure determined to date, and allows the comparison of the structures of recombinant Fe(III)-hTF/2N expressed in P. pastoris and mammalian cells with serum-derived transferrin. The polypeptide folding pattern is essentially identical in all of the three proteins. Mass spectroscopic analyses of P. pastoris- hTF/2N and proteolytically derived fragments revealed glycosylation of Ser-32 with a single hexose. This represents the first localization of an O-linked glycan in a P. pastoris-derived protein. Because of its distance from the iron-binding site, glycosylation of Ser-32 should not affect the iron-binding properties of hTF/2N expressed in P. pastoris, making this an excellent expression system for the production of hTF/2N.     

Mutational analysis of C-lobe ligands of human serum transferrin: insights into the mechanism of iron release

Each homologous lobe of human serum transferrin (hTF) has one Fe(3+) ion bound by an aspartic acid, a histidine, two tyrosine residues, and two oxygens from the synergistic anion, carbonate. Extensive characterization of these ligands in the N-terminal lobe has been carried out. Despite sharing the same set of ligands, there is a substantial amount of evidence that the N- and C-lobes are inequivalent. Studies of full-length hTF have shown that iron release from each lobe is kinetically distinguishable. To simplify the assessment of mutations in the C-lobe, we have created mutant hTF molecules in which the N-lobe binds iron with high affinity or not at all. Mutations targeting the C-lobe liganding residues have been introduced into these hTF constructs. UV-visible spectral, kinetic, and EPR studies have been undertaken to assess the effects of each mutation and to allow direct comparison to the N-lobe. As found for the N-lobe, the presence of Y517 in the C-lobe (equivalent to Y188 in the N-lobe) is absolutely essential for the binding of iron. Unlike the N-lobe, however, mutation of Y426 (equivalent to Y95) does not produce a stable complex with iron. For the mutants that retain the ability to bind iron (D392S and H585A), the rates of release are considerably slower than those measured for equivalent mutations in the N-lobe at both pH 7.4 and pH 5.6. Equilibrium binding experiments with HeLa S(3) cells indicate that recombinant hTF, in which Y426 or H585 is mutated, favor a closed or nearly closed conformation while those with mutations of the D392 or Y517 ligands appear to promote an open conformation. The differences in the effects of mutating the liganding residues in the two lobes and the subtle indications of cooperativity between lobes point to the importance of the transferrin receptor in effecting iron release from the C-lobe. Significantly, the equilibrium binding experiments also indicate that, regardless of which lobe contains the iron, the free energy of binding is equivalent and not additive; each monoferric hTF has a free energy of binding that is 82% of diferric hTF.     

Investigation of the mechanism of iron release from the C-lobe of human serum transferrin: mutational analysis of the role of a pH sensitive triad

The transferrins (TFs) are a family of proteins that are widely distributed in vertebrates, where they serve a major role in iron binding and transport. Most TFs are composed of two homologous lobes, the N- and C-lobes, each able to bind a single iron atom. Human serum transferrin (hTF) binds iron in the blood and delivers it to actively dividing cells; through the process of receptor-mediated endocytosis, diferric hTF in the serum (pH approximately 7.4) binds to specific TF receptors on the cell surface and is internalized, whereupon a pH drop in the endosome (pH approximately 5.6) facilitates iron release. Many factors affect the rate of iron release, including pH, chelator, temperature, salt, and lobe-lobe interactions. We, and others, have actively studied the mechanism of iron release from the recombinant N-lobe of hTF; in contrast, the exact details of iron release from the C-lobe have remained less well characterized but appear to differ from those found for the N-lobe. Recently, to simplify the purification protocol, we have expressed and purified full-length recombinant hTF containing an N-terminal hexahistidine tag [Mason et al. (2002) Biochemistry 41, 9448-9454]. In the present work, we have expressed a full-length recombinant hTF containing a K206E mutation such that the N-lobe does not readily release iron. The resulting full-length hTF allows us to focus on the C-lobe and to study the effects of mutations introduced into the C-lobe. The success of this strategy is documented and in vitro mutagenesis is used to identify three residues in the C-lobe that are critical for iron-release. Although the importance of this triad is unequivocally demonstrated, further studies are needed to completely elucidate the mechanism of iron release from the C-lobe of hTF. In addition, the striking difference in the effect of increasing salt concentrations on iron release from the two lobes of hTF is further documented in the present work.     

Competitive binding of bismuth to transferrin and albumin in aqueous solution and in blood plasma

Several bismuth compounds are currently used as antiulcer drugs, but their mechanism of action is not well established. Proteins are thought to be target sites. In this work we establish that the competitive binding of Bi(3+) to the blood serum proteins albumin and transferrin, as isolated proteins and in blood plasma, can be monitored via observation of (1)H and (13)C NMR resonances of isotopically labeled [epsilon-(13)C]Met transferrin. We show that Met(132) in the I132M recombinant N-lobe transferrin mutant is a sensitive indicator of N-lobe metal binding. Bi(3+) binds to the specific Fe(3+) sites of transferrin and the observed shifts of Met resonances suggest that Bi(3+) induces similar conformational changes in the N-lobe of transferrin in aqueous solution and plasma. Bi(3+) binding to albumin is nonspecific and Cys(34) is not a major binding site, which is surprising because Bi(3+) has a high affinity for thiolate sulfur. This illustrates that the potential target sites for metals (in this case Bi(3+)) in proteins depend not only on their presence but also on their accessibility. Bi(3+) binds to transferrin in preference to albumin both in aqueous solution and in blood plasma.     

Interlobe communication in human serum transferrin: metal binding and conformational dynamics investigated by electrospray ionization mass spectrometry

Human serum transferrin (hTF) is an iron transport protein, comprising two lobes (N and C), each containing a single metal-binding center. Despite substantial structural similarity between the two lobes, studies have demonstrated the existence of significant differences in their metal-binding properties. The nature of these differences has been elucidated through the use of electrospray ionization mass spectrometry to study both metal retention and conformational properties of hTF under a variety of conditions. In the absence of chelating agents or nonsynergistic anions, the diferric form of hTF remains intact until the pH is lowered to 4.5. The monoferric form of hTF retains the compact conformation until the pH is lowered to 4.0, whereas the apoprotein becomes partially unfolded at pH as high as 5.5. Selective (lobe-specific) modulation of the iron-binding properties of hTF using recombinant forms of the protein (in which the pH-sensitive elements in each lobe were mutated) verifies that the N-lobe of the protein has a lower affinity for ferric ion. Surprisingly, the apo-N-lobe is significantly less flexible compared to the apo-C-lobe. Furthermore, the conformation of the iron-free N-lobe is stabilized when the C-lobe contains iron, confirming the existence of an interlobe interaction within the protein. The experimental results provide strong support for the earlier suggestion that hTF interacts with its receptor (TFR) primarily through the C-lobe both at the cell surface and inside the endosome.     

Structure-based mutagenesis reveals critical residues in the transferrin receptor participating in the mechanism of pH-induced release of iron from human serum transferrin

The recent crystal structure of two monoferric human serum transferrin (Fe(N)hTF) molecules bound to the soluble portion of the homodimeric transferrin receptor (sTFR) has provided new details about this binding interaction that dictates the delivery of iron to cells. Specifically, substantial rearrangements in the homodimer interface of the sTFR occur as a result of the binding of the two Fe(N)hTF molecules. Mutagenesis of selected residues in the sTFR highlighted in the structure was undertaken to evaluate the effect on function. Elimination of Ca(2+) binding in the sTFR by mutating two of four coordinating residues ([E465A,E468A]) results in low production of an unstable and aggregated sTFR. Mutagenesis of two histidines ([H475A,H684A]) at the dimer interface had little effect on the kinetics of release of iron at pH 5.6 from either lobe, reflecting the inaccessibility of this cluster to solvent. Creation of an H318A sTFR mutant allows assignment of a small pH-dependent initial decrease in the magnitude of the fluorescence signal to His318. Removal of the four C-terminal residues of the sTFR, Asp757-Asn758-Glu759-Phe760, eliminates pH-stimulated release of iron from the C-lobe of the Fe(2)hTF/sTFR Δ757-760 complex. The inability of this sTFR mutant to bind and stabilize protonated hTF His349 (a pH-inducible switch) in the C-lobe of hTF accounts for the loss. Collectively, these studies support a model in which a series of pH-induced events involving both TFR residue His318 and hTF residue His349 occurs to promote receptor-stimulated release of iron from the C-lobe of hTF.     

Human serum transferrin: a tale of two lobes. Urea gel and steady state fluorescence analysis of recombinant transferrins as a function of pH, time, and the soluble portion of the transferrin receptor

Iron release from human serum transferrin (hTF) has been studied extensively; however, the molecular details of the mechanism(s) remain incomplete. This is in part due to the complexity of this process, which is influenced by lobe–lobe interactions, the transferrin receptor (TFR), the salt effect, the presence of a chelator, and acidification within the endosome, resulting in iron release. The present work brings together many of the concepts and assertions derived from previous studies in a methodical, uniform, and visual manner. Examination of earlier work reveals some uncertainty due to sample and technical limitations. We have used a combination of steady-state fluorescence and urea gels to evaluate the effect of conformation, pH, time, and the soluble portion of the TFR (sTFR) on iron release from each lobe of hTF. The use of authentic recombinant monoferric and locked species removes any possibility of cross-contamination by acquisition of iron. Elimination of detergent by use of the sTFR provides a further technical advantage. We find that iron release from the N-lobe is very sensitive to the conformation of the C-lobe, but is insensitive to the presence of the sTFR or to changes in pH (between 5.6 and 6.4). Specifically, when the cleft of the C-lobe is locked, the urea gels indicate that only about half of the iron is completely removed from the cleft of the N-lobe. Iron release from the C-lobe is most affected by the presence of the sTFR and changes in pH, but is unaffected by the conformation of the N-lobe. A model for iron release from diferric hTF is provided to delineate our findings. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Ionic residues of human serum transferrin affect binding to the transferrin receptor and iron release

Efficient delivery of iron is critically dependent on the binding of diferric human serum transferrin (hTF) to its specific receptor (TFR) on the surface of actively dividing cells. Internalization of the complex into an endosome precedes iron removal. The return of hTF to the blood to continue the iron delivery cycle relies on the maintenance of the interaction between apohTF and the TFR after exposure to endosomal pH (≤6.0). Identification of the specific residues accounting for the pH-sensitive nanomolar affinity with which hTF binds to TFR throughout the cycle is important to fully understand the iron delivery process. Alanine substitution of 11 charged hTF residues identified by available structures and modeling studies allowed evaluation of the role of each in (1) binding of hTF to the TFR and (2) TFR-mediated iron release. Six hTF mutants (R50A, R352A, D356A, E357A, E367A, and K511A) competed poorly with biotinylated diferric hTF for binding to TFR. In particular, we show that Asp356 in the C-lobe of hTF is essential to the formation of a stable hTF-TFR complex: mutation of Asp356 in the monoferric C-lobe hTF background prevented the formation of the stoichiometric 2:2 (hTF:TFR monomer) complex. Moreover, mutation of three residues (Asp356, Glu367, and Lys511), whether in the diferric or monoferric C-lobe hTF, significantly affected iron release when in complex with the TFR. Thus, mutagenesis of charged hTF residues has allowed identification of a number of residues that are critical to formation of and release of iron from the hTF-TFR complex.     

Effect of glycosylation on the function of a soluble, recombinant form of the transferrin receptor

Production of the soluble portion of the transferrin receptor (sTFR) by baby hamster kidney (BHK) cells is described, and the effect of glycosylation on the biological function of sTFR is evaluated for the first time. The sTFR (residues 121-760) has three N-linked glycosylation sites (Asn251, Asn317, and Asn727). Although fully glycosylated sTFR is secreted into the tissue culture medium ( approximately 40 mg/L), no nonglycosylated sTFR could be produced, suggesting that carbohydrate is critical to the folding, stability, and/or secretion of the receptor. Mutants in which glycosylation at positions 251 and 727 (N251D and N727D) is eliminated are well expressed, whereas production of the N317D mutant is poor. Analysis by electrospray ionization mass spectrometry confirms dimerization of the sTFR and the absence of the carbohydrate at the single site in each mutant. The effect of glycosylation on binding to diferric human transferrin (Fe(2) hTF), an authentic monoferric hTF with iron in the C-lobe (designated Fe(C) hTF), and a mutant (designated Mut-Fe(C) hTF that features a 30-fold slower iron release rate) was determined by surface plasmon resonance; a small ( approximately 20%) but consistent difference is noted for the binding of Fe(C) hTF and the Mut-Fe(C) hTF to the sTFR N317D mutant. The rate of iron release from Fe(C) hTF and Mut-Fe(C) hTF in complex with the sTFR and the sTFR mutants at pH 5.6 reveals that only the N317D mutant has a significant effect. The carbohydrate at position 317 lies close to a region of the TFR previously shown to interact with hTF.     

Sites on calmodulin that interact with the C-terminal tail of Cav1.2 channel

Two fragments of the C-terminal tail of the alpha(1) subunit (CT1, amino acids 1538-1692 and CT2, amino acids 1596-1692) of human cardiac L-type calcium channel (Ca(V)1.2) have been expressed, refolded, and purified. A single Ca(2+)-calmodulin binds to each fragment, and this interaction with Ca(2+)-calmodulin is required for proper folding of the fragment. Ca(2+)-calmodulin, bound to these fragments, is in a more extended conformation than calmodulin bound to a synthetic peptide representing the IQ motif, suggesting that either the conformation of the IQ sequence is different in the context of the longer fragment, or other sequences within CT2 contribute to the binding of calmodulin. NMR amide chemical shift perturbation mapping shows the backbone conformation of calmodulin is nearly identical when bound to CT1 and CT2, suggesting that amino acids 1538-1595 do not contribute to or alter calmodulin binding to amino acids 1596-1692 of Ca(V)1.2. The interaction with CT2 produces the greatest changes in the backbone amides of hydrophobic residues in the N-lobe and hydrophilic residues in the C-lobe of calmodulin and has a greater effect on residues located in Ca(2+) binding loops I and II in the N-lobe relative to loops III and IV in the C-lobe. In conclusion, Ca(2+)-calmodulin assumes a novel conformation when part of a complex with the C-terminal tail of the Ca(V)1.2 alpha(1) subunit that is not duplicated by synthetic peptides corresponding to the putative binding motifs.     

Photogeneration of titanium(III) from titanium(IV) citrate in aqueous solution

Current interest in the biochemistry of Ti(IV) arises from its widespread use in white pigments and its potential in therapeutic agents. Citrate is known to form strong complexes with Ti(IV). We show here that Ti(III) citrate is generated in a facile manner and in good yield by the action of UV radiation on Ti(IV) citrate in aqueous solution. The Ti(III)-citrate species formed was isolated and characterised by UV-Visible spectroscopy, showing an absorption at 547 nm (epsilon=100 M(-1)cm(-1)), and by electron paramagnetic resonance (EPR) spectroscopy giving a resonance at g=1.949 (linewidth=60G) . An X-ray structure of the parent Ti(IV) complex in the form [TiNa(3)(C(6)H(6)O(7))(2)(C(6)H(5)O(7))(H(2)O)(6.8)].2H(2)O is reported along with a study of the reaction of Ti(IV)-citrate with N,N-ethylenebis(o-hydroxytoluene)glycine (EHTG), which was more rapid than those of other related Ti(IV) complexes.     

Toxic and Physiological Metal Uptake and Release by Human Serum Transferrin

An atomistic understanding of metal transport in the human body is critical to anticipate the side effects of metal-based therapeutics and holds promise for new drugs and drug delivery designs. Human serum transferrin (hTF) is a central part of the transport processes because of its ubiquitous ferrying of physiological Fe(III) and other transition metals to tightly controlled parts of the body. There is an atomistic mechanism for the uptake process with Fe(III), but not for the release process, or for other metals. This study provides initial insight into these processes for a range of transition metals—Ti(IV), Co(III), Fe(III), Ga(III), Cr(III), Fe(II), Zn(II)—through fully atomistic, extensive quantum mechanical/discrete molecular dynamics sampling and provides, to our knowledge, a new technique we developed to calculate relative binding affinities between metal cations and the protein. It identifies protonation of Tyr188 as a trigger for metal release rather than protonation of Lys206 or Lys296. The study identifies the difficulty of metal release from hTF as potentially related to cytotoxicity. Simulations identify a few critical interactions that stabilize the metal binding site in a flexible, nuanced manner.     

Structure of the human transferrin receptor-transferrin complex

Iron, insoluble as free Fe(3+) and toxic as free Fe(2+), is distributed through the body as Fe(3+) bound to transferrin (Tf) for delivery to cells by endocytosis of its complex with transferrin receptor (TfR). Although much is understood of the transferrin endocytotic cycle, little has been uncovered of the molecular details underlying the formation of the receptor-transferrin complex. Using cryo-electron microscopy, we have produced a density map of the TfR-Tf complex at subnanometer resolution. An atomic model, obtained by fitting crystal structures of diferric Tf and the receptor ectodomain into the map, shows that the Tf N-lobe is sandwiched between the membrane and the TfR ectodomain and that the C-lobe abuts the receptor helical domain. When Tf binds receptor, its N-lobe moves by about 9 A with respect to its C-lobe. The structure of TfR-Tf complex helps account for known differences in the iron-release properties of free and receptor bound Tf.     

Chemical nature of implant-derived titanium(IV) ions in synovial fluid

Previous investigations have indicated a deleterious leakage of Ti(III) and/or Ti(IV) species from Ti-Al-V alloy joint prostheses into adjacent tissue, synovium or synovial fluid (SF) in vivo. In view of the importance of the particular chemical nature of such complexes in determining their biological activity, we have employed high field proton (1H) NMR spectroscopy to "speciate" Ti(IV) in inflammatory SF. Treatment of osteoarthritic SF samples with increasing concentrations of Ti(IV) (0.10-1.03 mM [TiO(C2O4)2]2-) gave rise to a specific broadening of the citrate proton resonances, indicating that this bioavailable oxygen-donor ligand plays an important role in complexing implant-derived Ti(IV). 1H NMR analysis of Ti(IV)-loaded SF samples subsequently treated with a large excess of ascorbate (0.05 M) showed that this added Ti(IV) chelator was only poorly effective in removing this metal ion from Ti(IV)-citrate/Ti(IV)-oxycitrate complexes. The results obtained here provide evidence for complexation of the low-molecular-mass (non-protein-bound) fraction of implant-derived Ti(IV) by citrate in vivo.     

Anion binding by human lactoferrin: results from crystallographic and physicochemical studies.

The anion-binding properties of lactoferrin (Lf), with Fe3+ or Cu2+ as the associated metal ion, have been investigated by physicochemical and crystallographic techniques. These highlight differences between the two sites and in the anion-binding behavior when different metals are bound. Carbonate, oxalate, and hybrid carbonate-oxalate complexes have been prepared and their characteristic electronic and EPR spectra recorded. Oxalate can displace carbonate from either one or both anion sites of Cu2(CO3)2Lf, depending on the oxalate concentration, but no such displacement occurs for Fe2(CO3)2Lf. Addition of oxalate and the appropriate metal ion to apoLf under carbonate-free conditions gives dioxalate complexes with both Fe3+ and Cu2+, except when traces of EDTA remain associated with the protein, when hybrid complexes M2(CO3)(C2O4)Lf can result. The anion sites in the crystal structures of Fe2(CO3)2Lf, Cu2-(CO3)2Lf, and Cu2(CO3)(C2O4)Lf, refined at 2.2, 2.1, and 2.2 A, respectively, have been compared. In every case, the anion is hydrogen bonded to the N-terminus of helix 5, an associated arginine side chain, and a nearby threonine side chain. The carbonate ion binds in bidentate fashion to the metal, except in the N-lobe site of dicupric lactoferrin, where it is monodentate; the difference arises from slight movement of the metal ion. The hybrid complex shows that the oxalate ion binds preferentially in the C-lobe site, in 1,2-bidentate mode, but with the displacement of several nearby side chains. These observations lead to a generalized model for synergistic anion binding by transferrins.     

Histidine pK(a) values for the N-lobe of human transferrin: effect of substitution of binding site Asp by Ser (D63S)

The pK(a) values have been determined for eight of the nine histidine residues and the amino terminus of the N-lobe of human apo-transferrin (hTF/2N), and for seven of the nine histidine residues and the amino terminus of the protein Asp63Ser hTF/2N containing a mutation of the Fe(3+)-ligand Asp63 to Ser63. Calculations suggested that substitution of aspartate by serine would result in decreases of the pK(a) values of most of the histidine residues in the protein. This was found to be the case experimentally, and allowed assignment of the varepsilonCH resonance of His249. For the wild-type protein, the His residue with a pK(a) of 7.40 was assigned as His249, whereas for the mutant, no observable His residue had a pK(a) value higher than 6.9. The protonated form of His249 appears to be stabilised by interactions with Asp63, and the high pK(a) value may be critical for ensuring the release of iron at endosomal pH (5.5). The mutation lowered the apparent binding constant of hTF/2N for the synergistic anion oxalate from log K 4.0 to log K 3.3. (1)H NMR spectral changes induced by Ga(3+) binding to the mutant are compared to those observed for the wild-type protein.     

Iron-binding process in the amino- and carboxyl-terminal lobes of ovotransferrin: quantitative studies utilizing single Fe3+-binding mutants

Iron-liganding-residue mutants of ovotransferrin, Y191F and Y524F, were investigated for their Fe(3+)-binding properties. The absorption spectrum and urea gel electrophoresis verified the single iron binding on the C- and N-lobes for Y191F and Y524F, respectively. A newly developed competitive Fe(3+)-binding analysis, in which equimolar Y191F and Y524F are mixed with less Fe(3+) than saturation, enabled us to quantitatively determine the lobe preference for initial iron entry as the ratio (alpha value) of N-lobe over C-lobe. The alpha value estimated on the basis of a kinetic model was highly dependent on pH; within a pH range from 6.5 to 9.0, alpha was increased from 2 to 5 on lowering pH with an apparent sigmoid curve. On differential scanning calorimetry, single thermal transition was observed around 61 degrees C for the apo forms of Y191F, Y524F, and wild-type ovotransferrin. The Fe(3+)-loaded mutants, however, showed dual transitions at 62.4 and 82.1 degrees C in Y191F and 66.4 and 76.0 degrees C in Y524F. According to the DeltaG(AB) value that is defined as the free energy change in a target lobe induced by the iron binding on the counter lobe, marked stabilization effects by interlobe interactions were found to be induced during the major iron-binding process: upon the primary N-lobe iron binding in the iron-free C-lobe (DeltaG(AB), -2.25 kcal/mol) and upon the secondary C-lobe iron binding in the monoferric N-lobe (DeltaG(AB), -6.45 kcal/mol).     

Formation of titanium(IV) transferrin by reaction of human serum apotransferrin with titanium complexes

The reaction of human serum apotransferrin with titanium(IV) citrate under physiological conditions results in the formation of a specific bis-titanium(IV) transferrin adduct (Ti₂Tf hereafter) with two titanium(IV) ions loaded at the iron binding sites. The same specific Ti₂Tf complex is formed by reacting apotransferrin with titanium(III) chloride and exposing the sample to air. The derivative thus obtained was characterized by spectroscopic techniques, including absorption, UV difference, circular dichroism and ¹³C NMR spectroscopies, and shown to be stable within the pH range 5.5–9.0. Surprisingly, the reaction of apoTf with titanium(IV) nitrilotriacetate (NTA) does not lead to formation of appreciable amounts of Ti₂Tf, even after long incubation times, although some weak interactions of Ti(IV)NTA with apoTf are spectroscopically detected. Implications of the present results for a role of transferrin in the uptake, transport and delivery of soluble titanium(IV) compounds under physiological conditions are discussed.     

The oxalate effect on release of iron from human serum transferrin explained

A unique feature of the mechanism of iron binding to the transferrin (TF) family is the synergistic relationship between metal binding and anion binding. Little or no iron will bind to the protein without concomitant binding of an anion, physiologically identified as carbonate. Substitution of oxalate for carbonate produces no significant changes in polypeptide folding or domain orientation in the N-lobe of human serum TF (hTF) as revealed by our 1.2A structure. The oxalate is able to bind to the iron in a symmetric bidentate fashion, which, combined with the low pK(a) of the oxalate anion, makes iron displacement more difficult as documented by both iron release kinetic and equilibrium data. Characterization of an N-lobe in which the arginine at position 124 is mutated to alanine reveals that the stabilizing effect of oxalate is even greater in this mutant and nearly cancels the destabilizing effect of the mutation. Importantly, incorporation of oxalate as the synergistic anion appears to completely inhibit removal of iron from recombinant full-length hTF by HeLa S(3) cells, strongly indicating that oxalate also replaces carbonate in the C-lobe to form a stable complex. Kinetic studies confirm this claim. The combination of structural and functional data provides a coherent delineation of the effect of oxalate binding on hTF and rationalizes the results of many previous studies. In the context of iron uptake by cells, substitution of carbonate by oxalate effectively locks the iron into each lobe of hTF, thereby interfering with normal iron metabolism.