Worldwide stocks of actinides and lanthanide fission products produced through conventional nuclear spent fuel are increasing continuously, resulting in a growing risk of environmental and human exposure to these toxic radioactive metal ions. Understanding the bio-molecular pathways involved in mammalian uptake, transport and storage of these f-elements is crucial to the development of new decontamination strategies and could also be beneficial to the design of new containment and separation processes. To start unraveling these pathways, our approach takes advantage of the unique spectroscopic properties of trivalent curium. We clearly show that the human iron transporter transferrin acts as an antenna that sensitizes curium luminescence through intramolecular energy transfer. This behavior has been used to describe the coordination of curium within the two binding sites of the protein and to investigate the recognition of curium-transferrin complexes by the cognate transferrin receptor. In addition to providing the first protein-curium spectroscopic characterization, these studies prove that transferrin receptor-mediated endocytosis is a viable mechanism of intracellular entry for trivalent actinides such as curium and provide a new tool utilizing the specific luminescence of curium for the determination of other biological actinide transport mechanisms.
The Fe3+ binding protein human serum transferrin (hTF) is well known for its role in cellular iron delivery via the transferrin receptor (TFR). A new application is the use of hTF as a therapy and targeted drug delivery system for a number of diseases. Recently, production of hTF in plants has been reported; such systems provide a relatively inexpensive, animal-free (eliminating potential contamination by animal pathogens) method to produce large amounts of recombinant proteins for such biopharmaceutical applications. Specifically, the production of Optiferrin™ (hTF produced in rice, Oryza sativa, from InVitria) has been shown to yield large amounts of functional protein for use in culture medium for cellular iron delivery to promote growth. In the present work we describe further purification (by gel filtration) and characterization of hTF produced in rice (purified Optiferrin™) to determine its suitability in biopharmaceutical applications. The spectral, mass spectrometric, urea gel and kinetic analysis shows that purified Optiferrin™ is similar to recombinant nonglycosylated N-His tagged hTF expressed by baby hamster kidney cells and/or serum derived glycosylated hTF. Additionally, in a competitive immunoassay, iron-loaded Optiferrin™ is equivalent to iron-loaded N-His hTF in its ability to bind to the soluble portion of the TFR immobilized in an assay plate. As an essential requirement for any functional hTF, both lobes of purified Optiferrin™ bind Fe3+ tightly yet reversibly. Although previously shown to be capable of delivering Fe3+ to cells, the kinetics of iron release from iron-loaded Optiferrin™/sTFR and iron-loaded N-His hTF/sTFR complexes differ somewhat. We conclude that the purified Optiferrin™ might be suitable for consideration in biopharmaceutical applications.
human serum transferrin; Optiferrin™; recombinant transferrin; mass spectrometry; iron delivery; kinetics; transferrin receptor
The recent crystal structure of two monoferric human serum transferrin (FeNhTF) molecules bound to the soluble portion of the homodimeric transferrin receptor (sTFR) has provided new details of this binding interaction which dictates iron delivery to cells. Specifically, substantial rearrangements in the homodimer interface of the sTFR occur as a result of the binding of the two FeNhTF molecules. Mutagenesis of selected residues in the sTFR highlighted in the structure was undertaken to evaluate the effect on function. Elimination of Ca2+ 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 iron release at pH 5.6 from either lobe, reflecting the inaccessibility of this cluster to solvent. Creation of a H318A sTFR mutant allows assignment of a small pH dependent initial decrease in the fluorescent signal to His318. Removal of the four C-terminal residues of the sTFR, Asp757-Asn758-Glu759-Phe760, eliminates pH-stimulated iron release from the C-lobe of the Fe2hTF/sTFR Δ757–760 complex. The loss is accounted for by the inability of this sTFR mutant to bind and stabilize protonated hTF His349 (a pH-inducible switch) in the C-lobe of hTF. Collectively, these studies support a model in which a series of pH-induced events involving both TFR residue His318 and hTF residue His349 occurs in order to promote receptor-stimulated iron release from the C-lobe of hTF.
Human serum transferrin (hTF) is a bilobal glycoprotein that reversibly binds Fe3+ and delivers it to cells by the process of receptor-mediated endocytosis. Despite decades of research, the precise events resulting in iron release from each lobe of hTF within the endosome have not been fully delineated.
Scope of Review
We provide an overview of the kinetics of iron release from hTF ± the transferrin receptor (TFR) at endosomal pH (5.6). A critical evaluation of the array of biophysical techniques used to determine accurate rate constants is provided.
Delivery of Fe3+ by to actively dividing cells by hTF is essential; too much or too little Fe3+ directly impacts the well-being of an individual. Because the interaction of hTF with the TFR controls iron distribution in the body, an understanding of this process at the molecular level is essential.
Not only does TFR direct the delivery of iron to the cell through the binding of hTF, kinetic data demonstrate that it also modulates iron release from the N- and C-lobes of hTF. Specifically, the TFR balances the rate of iron release from each lobe, resulting in efficient Fe3+ release within a physiologically relevant time frame.
transferrin; transferrin receptor; kinetics; fluorescence
Not long after the Big Bang, iron began to play a central role in the Universe and soon became mired in the tangle of biochemistry that is the prima essentia of life. Since life’s addiction to iron transcends the oxygenation of the Earth’s atmosphere, living things must be protected from the potentially dangerous mix of iron and oxygen. The human being possesses grams of this potentially toxic transition metal, which is shuttling through his oxygen-rich humor. Since long before the birth of modern medicine, the blood—vibrant red from a massive abundance of hemoglobin iron—has been a focus for health experts.
Scope of Review
We describe the current understanding of iron metabolism, highlight the many important discoveries that accreted this knowledge, and describe the perils of dysfunctional iron handling.
Isaac Newton famously penned, “If I have seen further than others, it is by standing upon the shoulders of giants”. We hope that this review will inspire future scientists to develop intellectual pursuits by understanding the research and ideas from many remarkable thinkers of the past.
The history of iron research is a long, rich story with early beginnings, and is far from being finished.
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 eleven 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) in 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 iron release from the hTF/TFR complex.
The transferrins are a family of bilobal iron-binding proteins that play the crucial role of binding ferric iron and keeping it in solution, thereby controlling the levels of this important metal. Human serum transferrin (hTF) carries one iron in each of two similar lobes. Understanding the detailed mechanism of iron release from each lobe of hTF during receptor mediated endocytosis has been extremely challenging because of the active participation of the transferrin receptor (TFR), salt, a chelator, lobe-lobe interactions and the low pH within the endosome. Our use of authentic monoferric hTF (unable to bind iron in one lobe) or of diferric hTF (with iron locked in one lobe), provided distinct kinetic end points allowing us to bypass many of the previous difficulties. The capture and unambiguous assignment of all kinetic events associated with iron release by stopped flow spectrofluorimetry, in the presence and absence of the TFR, unequivocally establishes the decisive role of the TFR in promoting efficient and balanced iron release from both lobes of hTF during one endocytic cycle. For the first time the four microscopic rate constants required to accurately describe the kinetics of iron removal are reported for hTF with and without the TFR. Specifically, at pH 5.6, the TFR enhances the rate of iron release from the C-lobe (7- to 11-fold), and slows the rate of iron release from the N-lobe (6- to 15-fold), making them more equivalent and producing an increase in the net rate of iron removal from Fe2hTF. Calculated cooperativity factors, in addition to plots of time dependent species distributions in the absence and presence of the TFR clearly illustrate the differences. Accurate rate constants for the pH and salt induced conformational changes in each lobe precisely delineate how delivery of iron within the physiologically relevant time frame of 2 min might be accomplished.
Stopped flow fluorescence; iron release kinetics; salt effect; iron release model
Human serum transferrin (hTF) is a bilobal glycoprotein that transports iron to cells. At neutral pH, diferric hTF binds with nM affinity to the transferrin receptor (TFR) on the cell surface. The complex is taken into the cell where, at the acidic pH of the endosome (~pH 5.6), iron is released. Since iron coordination strongly quenches the intrinsic tryptophan fluorescence of hTF, the increase in the fluorescent signal reports the rate constant(s) of iron release. At pH 5.6, the TFR considerably enhances iron release from the C-lobe (with little effect on iron release from the N-lobe). The recombinant soluble TFR is a dimer with 11 tryptophan residues per monomer. In the hTF/TFR complex these residues could contribute to and compromise the readout ascribed to iron release from hTF. We report that compared to FeC hTF alone, the increase in the fluorescent signal from the preformed complex of FeC hTF and the TFR at pH 5.6 is significantly quenched (75%). To dissect the contributions of hTF and the TFR to the change in fluorescence, 5-hydroxytryptophan was incorporated into each using our mammalian expression system. Selective excitation of the samples at 280 or 315 nm shows that the TFR contributes little or nothing to the increase in fluorescence when ferric iron is released from FeC hTF. Quantum yield determinations of TFR, FeC hTF and the FeC hTF/TFR complex strongly support our interpretation of the kinetic data.
Metalloproteins; protein-receptor interaction; tryptophan analogues; tryptophan fluorescence; stopped-flow kinetics; BHK cells
Human serum transferrin (hTF) binds two ferric iron ions which are delivered to cells in a transferrin receptor (TFR) mediated process. Critical to the delivery of iron to cells is the binding of hTF to the TFR and the efficient release of iron orchestrated by the interaction. Within the endosome, iron release from hTF is also aided by lower pH, the presence of anions, and a chelator yet to be identified. We have recently shown that three of the four residues comprising a loop in the N-lobe (Pro142, Lys144, and Pro145) are critical to the high-affinity interaction of hTF with the TFR. In contrast, Arg143 in this loop does not participate in the binding isotherm. In the current study, the kinetics of iron release from alanine mutants of each of these four residues (placed into both diferric and monoferric N-lobe backgrounds) have been determined ± the TFR. The R143A mutation greatly retards the rate of iron release from the N-lobe in the absence of the TFR but has considerably less of an effect in its presence. Our data definitively show that Arg143 serves as a kinetically significant anion binding (KISAB) site that is, by definition, sensitive to salt concentration and critical to the conformational change necessary to induce iron release from the N-lobe of hTF (in the absence of the TFR). This is the first identification of an authentic KISAB site in the N-lobe of hTF. The effect of the single R143A mutation on the kinetic profile of iron release provides a dramatic illustration of the dynamic nature of hTF.
Essential to iron transport and delivery, human serum transferrin (hTF) is a bilobal glycoprotein capable of reversibly binding one ferric ion in each lobe (the N- and C-lobes). A complete description of iron release from hTF, as well as insight into the physiological significance of the bilobal structure, demands characterization of the isolated lobes. Although production of large amounts of isolated N-lobe and full-length hTF has been well documented, attempts to produce the C-lobe (by recombinant and/or proteolytic approaches) have met with more limited success. Our new strategy involves replacing the hepta-peptide, PEAPTDE (comprising the bridge between the lobes) with the sequence ENLYFQ/G in a His-tagged non-glycosylated monoferric hTF construct, designated FeChTF. The new bridge sequence of this construct, designated FeCTEV hTF, is readily cleaved by the tobacco etch virus (TEV) protease yielding non-glycosylated C-lobe. Following nickel column chromatography (to remove the N-lobe and the TEV protease which are both His tagged), the homogeneity of the C-lobe has been confirmed by mass spectroscopy. Differing reactivity with a monoclonal antibody specific to the C-lobe indicates that introduction of the TEV cleavage site into the bridge alters its conformation. The spectral and kinetic properties of the isolated C-lobe differ significantly from those of the isolated N-lobe.
Tobacco etch virus protease; transferrin; stopped-flow fluorescence; intrinsic tryptophan fluorescence; absorption coefficient determination; cooperativity
Glioblastoma multiforme (GBM) is the most common and lethal primary brain tumor with median survival of only 12-15 months under the current standard of care. To both increase tumor specificity and decrease nonspecific side effects, recent experimental strategies in the treatment of GBM have focused on targeting cell surface receptors, including the transferrin (Tf) receptor, that are overexpressed in many cancers. A major limitation of Tf-based therapeutics is the short association of Tf within the cell to deliver its payload. We previously developed two mutant Tf molecules, K206E/R632A Tf and K206E/K534A Tf, in which iron is locked into each of the two homologous lobes. Relative to wild-type Tf, we showed enhanced delivery of diphtheria toxin (DT) from these mutants to a monolayer culture of HeLa cells. Here, we extend the application of our Tf mutants to the treatment of GBM. In vitro treatment of Tf mutants to a monolayer culture of glioma cells demonstrated enhanced cellular association as well as enhanced delivery of conjugated DT. Treatment of GBM xenografts with mutant Tf conjugated DT resulted in pronounced regression in vivo, indicating their potential use as drug carriers.
Transferrin; Diphtheria toxin; Glioblastoma multiforme; Mouse model; Targeted therapy
Transferrin is the main iron transport protein found in the circulation, and the level of transferrin saturation in the blood is an important indicator of iron status. The peptides amidated gastrin17 (Gamide) and glycine-extended gastrin17 (Ggly) are well known for their roles in controlling acid secretion and as growth factors in the gastrointestinal tract. Several lines of evidence, including the facts that transferrin binds gastrin, that gastrins bind ferric ions, and that the level of expression of gastrins positively correlates with transferrin saturation, suggest the possible involvement of the transferrin-gastrin interaction in iron homeostasis. In the present work the interaction between gastrins and transferrin has been characterized by surface plasmon resonance and covalent crosslinking. Firstly, an interaction between iron-free apo-transferrin and Gamide or Ggly was observed. The fact that no interaction was observed in the presence of the chelator EDTA suggested that the gastrin-ferric ion complex was the interacting species. Moreover, removal of ferric ions with EDTA reduced the stability of the complex between apo-transferrin and gastrins, and no interaction was observed between Gamide or Ggly and diferric-transferrin. Secondly, some or all of glutamates at positions 8–10 of the Ggly molecule, together with the C-terminal domain, were necessary for the interaction with apo-transferrin. Thirdly, monoferric transferrin mutants incapable of binding iron in either the N- or C-terminal lobe still bound Ggly. These findings are consistent with the hypothesis that gastrin peptides bind to non-ligand residues within the open cleft in each lobe of transferrin and are involved in iron loading of transferrin in vivo
Ferric; gastrin; iron; transferrin
Human serum transferrin (hTF), with two Fe3+ binding lobes transports iron into cells. Diferric hTF preferentially binds to a specific receptor (TFR) on the surface of cells and the complex undergoes clathrin dependent receptor-mediated endocytosis. The clathrin-coated vesicle fuses with an endosome where the pH is lowered, facilitating iron release from hTF. On a biologically relevant timescale (2-3 min), the factors critical to iron release include pH, anions, a chelator and the interaction of hTF with the TFR. Previous work, in which the increase in the intrinsic fluorescence signal was used to monitor iron release from the hTF/TFR complex, established that the TFR significantly enhances the rate of iron release from the C-lobe of hTF. In the current study, the role of the five C-lobe Trp residues in reporting the fluorescence change has been evaluated (± sTFR). Only four of the five recombinant Trp→ Phe mutants produced well. A single slow rate constant for iron release is found for the monoferric C-lobe (FeC hTF) and the four Trp mutants in the FeC hTF background. The three Trp residues equivalent to those in the N-lobe differed from the N-lobe and each other in their contributions to the fluorescent signal. Two rate constants are observed for the FeC hTF control and the four Trp mutants in complex with the TFR: kobsC1 reports conformational change(s) in the C-lobe initiated by the TFR and kobsC2 is ascribed to iron release. Excitation at 295 nm (Trp only) and at 280 nm (Trp and Tyr) reveals interesting and significant differences in the rate constants for the complex.
The G65R mutation in the N-lobe of human transferrin was created to mimic a naturally occurring variant (G394R) found in the homologous C-lobe. Because Gly65 is hydrogen-bonded to the iron-binding ligand Asp63, it comprises part of the second shell hydrogen bond network surrounding the iron within the metal binding cleft of the protein. Substitution with an arginine residue at this position disrupts the network, resulting in much more facile removal of iron from the G65R mutant. As shown by UV-vis and EPR spectroscopy, and by kinetic assays measuring the release of iron, the G65R mutant can exist in three forms. Two of the forms (yellow and pink in color) are inter-convertible. The yellow form predominates in 1 M bicarbonate; the pink form is generated from the yellow form upon exchange into 1 M HEPES buffer, pH 7.4. The third form (also pink in color) is produced by the addition of Fe3+-(nitrilotriacetate)2 to apo-G65R. Hydrogen/deuterium exchange experiments are consistent with all forms of the G65R mutant assuming a more open conformation. Additionally, mass spectroscopic analysis reveals the presence of nitrilotriacetate in the third form. The inability to obtain crystals of the G65R mutant, led to development of a novel crystallization strategy in which the double mutation G65R/K206E stabilizes a single closed pink conformer and captures Arg65 in a single position. Collectively, these studies highlight the importance of the hydrogen bond network in the cleft, as well as the inherent flexibility of the N-lobe which although able to adapt to accommodate the large arginine substitution exists in multiple conformations.
We previously demonstrated that decreasing the iron release rate of transferrin (Tf), by replacing the synergistic anion carbonate with oxalate, increases its in vitro drug carrier efficacy in HeLa cells. In the current work, the utility of this strategy has been further explored by generating two Tf mutants, K206E/R632A Tf and K206E/K534A Tf, exhibiting different degrees of iron release inhibition. The intracellular trafficking behavior of these Tf mutants has been assessed by measuring their association with HeLa cells. Compared to native Tf, the cellular association of K206E/R632A Tf and K206E/K534A Tf increased by 126 and 250%, respectively. Surface plasmon resonance studies clearly indicate that this increase in cellular association is due to a decrease in the iron release rate and not to differences in binding affinity of the mutants to the Tf receptor (TfR). Diphtheria toxin (DT) conjugates of K206E/R632A Tf and K206E/K534A Tf showed significantly increased cytotoxicity against HeLa cells with IC50 values of 1.00 pM and 0.93 pM, respectively, compared to a value of 1.73 pM for the native Tf conjugate. Besides further validating our strategy of inhibiting iron release, these Tf mutants provide proof-of-principle that site-directed mutagenesis offers an alternative method for improving the drug carrier efficacy of Tf.
Transferrin; Diphtheria toxin; Site-directed mutagenesis; Intracellular trafficking; Targeted cancer therapy
The murine inhibitor of carbonic anhydrase (mICA) is a member of the superfamily related to the bilobal iron transport protein transferrin (TF), which binds a ferric ion within a cleft in each lobe. Although the gene encoding ICA in humans is classified as a pseudogene, an apparently functional ICA gene has been annotated in mice, rats, cows, pigs, and dogs. All ICAs lack one (or more) of the amino acid ligands in each lobe essential for high-affinity coordination of iron and the requisite synergistic anion, carbonate. The reason why ICA family members have lost the ability to bind iron is potentially related to acquiring a new function(s), one of which is inhibition of certain carbonic anhydrase (CA) isoforms. A recombinant mutant of the mICA (W124R/S188Y) was created with the goal of restoring the ligands required for both anion (Arg124) and iron (Tyr188) binding in the N-lobe. Absorption and fluorescence spectra definitively show that the mutant binds ferric iron in the N-lobe. Electrospray ionization mass spectrometry confirms the presence of both ferric iron and carbonate. At the putative endosomal pH of 5.6, iron is released by two slow processes indicative of high-affinity coordination. Induction of specific iron binding implies that (1) the structure of mICA resembles those of other TF family members and (2) the N-lobe can adopt a conformation in which the cleft closes when iron binds. Because the conformational change in the N-lobe indicated by metal binding does not impact the inhibitory activity of mICA, inhibition of CA was tentatively assigned to the C-lobe. Proof of this assignment is provided by limited trypsin proteolysis of porcine ICA.
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.
Cooperativity; Urea gels; Steady-state tryptophan fluorescence; Transferrin/transferrin receptor complex; Iron-release model
An accurate protein concentration is an essential component of most biochemical experiments. The simplest method to determine a protein concentration is by measuring the A280, using an absorption coefficient (ε), and applying the Beer-Lambert law. For some metalloproteins (including all transferrin family members) difficulties arise because metal binding contributes to the A280 in a non-linear manner. The Edelhoch method is based on the assumption that the ε of a denatured protein in 6 M guanidine-HCl can be calculated from the number of the tryptophan, tyrosine, and cystine residues. We extend this method to derive ε values for both apo- and iron-bound transferrins. The absorbance of an identical amount of iron containing protein is measured in: 1) 6 M guanidine-HCl (denatured, no iron); 2) pH 7.4 buffer (non-denatured with iron); and 3) pH 5.6 (or lower) buffer with a chelator (non-denatured without iron). Since the iron free apo-protein has an identical A280 under non-denaturing conditions, the difference between the reading at pH 7.4 and the lower pH directly reports the contribution of the iron. The method is fast and consumes ~1 mg of sample. The ability to determine accurate ε values for transferrin mutants that bind iron with a wide range of affinities has proven very useful; furthermore a similar approach could easily be followed to determine ε values for other metalloproteins in which metal binding contributes to the A280.
Transferrin; molar absorption coefficient; metalloproteins; Edelhoch method; ligand metal charge transfer
Transferrin (Tf) conjugates of CRM107 are currently being tested in clinical trials for treatment of malignant gliomas. However, the rapid cellular recycling of Tf limits its efficiency as a drug carrier. We have developed a mathematical model of the Tf/TfR trafficking cycle and have identified the Tf iron release rate as a previously unreported factor governing the degree of Tf cellular association. The release of iron from Tf is inhibited by replacing the synergistic carbonate anion with oxalate. Trafficking patterns for oxalate Tf and native Tf are compared by measuring their cellular association with HeLa cells. The amount of Tf associated with the cells is an average of 51% greater for oxalate Tf than for native Tf over a two hour period at Tf concentrations of 0.1 nM and 1 nM. Importantly, diphtheria toxin (DT) conjugates of oxalate Tf are more cytotoxic against HeLa cells than conjugates of native Tf. Conjugate IC50 values were determined to be 0.06 nM for the oxalate Tf conjugate vs. 0.22 nM for the native Tf conjugate. Thus, we show that inhibition of Tf iron release improves the efficacy of Tf as a drug carrier through increased association with cells expressing TfR.
Transferrin; Diphtheria toxin; Drug carrier; Trafficking; Targeted cancer therapy
Serum transferrin reversibly binds iron in each of two lobes and delivers it to cells by a receptor-mediated, pH-dependant process. The binding and release of iron results in a large conformational change in which two subdomains in each lobe close or open with a rigid twisting motion around a hinge. We report the structure of human serum transferrin (hTF) lacking iron (apo-hTF) which was independently determined by two methods: (1) the crystal structure of recombinant non-glycosylated apo-hTF was solved at 2.7 Å resolution using a MAD phasing strategy, by substituting the nine methionines in hTF with selenomethionine and (2) the structure of glycosylated apo-hTF (isolated from serum) was determined to a resolution of 2.7 Å by molecular replacement using the human apo-N-lobe and the rabbit holo-C1-subdomain as search models. These two crystal structures are essentially identical. They represent the first published model for full-length human TF and reveal that, in contrast to family members (human lactoferrin and hen ovotransferrin), both lobes are almost equally open: 59.4° and 49.5° rotations are required to open the N- and C-lobe, respectively, (compared to closed pig TF). Availability of this structure is critical to a complete understanding of the metal binding properties of each lobe of hTF; the apo-hTF structure suggests that differences in the hinge regions of the N- and C-lobes may influence the rates of iron binding and release. In addition, we evaluate potential interactions between apo-hTF and the human transferrin receptor.