A new method for obtaining human transferrin C-lobe in the native conformation: preparation and properties

Eukaryotic transferrins comprise a class of bilobal iron-binding proteins in which each lobe carries a single binding site. Although expression of full-length transferrins and their N-terminal lobes, in wild-type and mutated forms, has been successfully accomplished by several laboratories, expression of C-lobes has been much less satisfactory. A possible explanation of the difficulty is that proper folding of the C-lobe, with its 11 disulfide bonds, depends on prior synthesis and proper folding of the N-lobe. We have therefore developed a new strategy, introducing a specific factor Xa cleavage site in the interlobe-connecting strand to permit separation of the lobes after expression of the full-length protein. The resulting protein was expressed in satisfactory yield, >20 mg/L, and could be easily and completely cleaved to yield two distinguishable fragments representing N- and C-lobes, respectively. Retaining the glycosylation sites, found only in the C-lobe, made it possible to separate the fragments from each other by ConA affinity chromatography. The isolated C-lobe so obtained displayed spectroscopic and kinetic features of the C-lobe in native transferrin and was competent as an iron donor for K562 cells to which it bound in saturable fashion inhibitable by native diferric transferrin. Since the N-lobe by itself will neither bind nor donate iron to cells, the primary receptor-recognition site of transferrin resides in its C-lobe.     

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.     

An iron-dependent and transferrin-mediated cellular uptake pathway for plutonium

Plutonium is a toxic synthetic element with no natural biological function, but it is strongly retained by humans when ingested. Using small-angle X-ray scattering, receptor binding assays and synchrotron X-ray fluorescence microscopy, we find that rat adrenal gland (PC12) cells can acquire plutonium in vitro through the major iron acquisition pathway--receptor-mediated endocytosis of the iron transport protein serum transferrin; however, only one form of the plutonium-transferrin complex is active. Low-resolution solution models of plutonium-loaded transferrins derived from small-angle scattering show that only transferrin with plutonium bound in the protein's C-terminal lobe (C-lobe) and iron bound in the N-terminal lobe (N-lobe) (Pu(C)Fe(N)Tf) adopts the proper conformation for recognition by the transferrin receptor protein. Although the metal-binding site in each lobe contains the same donors in the same configuration and both lobes are similar, the differences between transferrin's two lobes act to restrict, but not eliminate, cellular Pu uptake.     

Camel lactoferrin, a transferrin-cum-lactoferrin: crystal structure of camel apolactoferrin at 2.6 A resolution and structural basis of its dual role

Camel lactoferrin is the first protein from the transferrin superfamily that has been found to display the characteristic functions of iron binding and release of lactoferrin as well as transferrin simultaneously. It was remarkable to observe a wide pH demarcation in the release of iron from two lobes. It loses 50 % iron at pH 6.5 and the remaining 50 % iron is released only at pH values between 4.0 and 2.0. Furthermore, proteolytically generated N and C-lobes of camel lactoferrin showed that the C-lobe lost iron at pH 6.5, while the N-lobe lost it only at pH less than 4.0. In order to establish the structural basis of this striking observation, the purified camel apolactoferrin was crystallized. The crystals belong to monoclinic space group C2 with unit cell dimensions a=175.8 A, b=80.9 A, c=56.4 A, beta=92.4 degrees and Z=4. The structure has been determined by the molecular replacement method and refined to an R-factor of 0.198 (R-free=0.268) using all the data in the resolution range of 20.0-2.6 A. The overall structure of camel apolactoferrin folds into two lobes which contain four distinct domains. Both lobes adopt open conformations indicating wide distances between the iron binding residues in the native iron-free form of lactoferrin. The dispositions of various residues of the iron binding pocket of the N-lobe of camel apolactoferrin are similar to those of the N-lobe in human apolactoferrin, while the corresponding residues in the C-lobe show a striking similarity with those in the C-lobes of duck and hen apo-ovotransferrins. These observations indicate that the N-lobe of camel apolactoferrin is structurally very similar to the N-lobe of human apolactoferrin and the structure of the C-lobe of camel apolactoferrin matches closely with those of the hen and duck apo-ovotransferrins. These observations suggest that the iron binding and releasing behaviour of the N-lobe of camel lactoferrin is similar to that of the N-lobe of human lactoferrin, whereas that of the C-lobe resembles those of the C-lobes of duck and hen apo-ovotransferrins. Hence, it correlates with the observation of the N-lobe of camel lactoferrin losing iron at a low pH (4.0-2.0) as in other lactoferrins. On the other hand, the C-lobe of camel lactoferrin loses iron at higher pH (7.0-6.0) like transferrins suggesting its functional similarity to that of transferrins. Thus, camel lactoferrin can be termed as half lactoferrin and half transferrin.     

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.     

The mechanism of iron release from the transferrin-receptor 1 adduct

We report the determination in cell-free assays of the mechanism of iron release from the N-lobe and C-lobe of human serum transferrin in interaction with intact transferrin receptor 1 at 4.3< or =pH< or =6.5. Iron is first released from the N-lobe in the tens of milliseconds range and then from the C-lobe in the hundreds of seconds range. In both cases, iron loss is rate-controlled by slow proton transfers, rate constant for the N-lobe k(1)=1.20(+/-0.05)x10(6)M(-1)s(-1) and for the C-lobe k(2)=1.6(+/-0.1)x10(3)M(-1)s(-1). This iron loss is subsequent to a fast proton-driven decarbonation and is followed by two proton gains, (pK(1a))/2=5.28 per proton for the N-lobe and (pK(2a))/2=5.10 per proton for the C-lobe. Under similar experimental conditions, iron loss is about 17-fold faster from the N-lobe and is at least 200-fold faster from the C-lobe when compared to holotransferrin in the absence of receptor 1. After iron release, the apotransferrin-receptor adduct undergoes a slow partial dissociation controlled by a change in the conformation of the receptor; rate constant k(3)=1.7(+/-0.1)x10(-3)s(-1). At endosomic pH, the final equilibrated state is attained in about 1000 s, after which the free apotransferrin, two prototropic species of the acidic form of the receptor and apotransferrin interacting with the receptor coexist simultaneously. However, since recycling of the vesicle containing the receptor to the cell surface takes a few minutes, the major part of transferrin will still be forwarded to the biological fluid in the form of the apotransferrin-receptor protein-protein adduct.     

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.     

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.     

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.     

Large cooperativity in the removal of iron from transferrin at physiological temperature and chloride ion concentration

Iron removal from serum transferrin by various chelators has been studied by gel electrophoresis, which allows direct quantitation of all four forms of transferrin (diferric, C-monoferric, N-monoferric, and apotransferrin). Large cooperativity between the two lobes of serum transferrin is found for iron removal by several different chelators near physiological conditions (pH 7.4, 37 °C, 150 mM NaCl, 20 mM NaHCO₃). This cooperativity is manifested in a dramatic decrease in the rate of iron removal from the N-monoferric transferrin as compared with iron removal from the other forms of ferric transferrin. Cooperativity is diminished as the pH is decreased; it is also very sensitive to changes in chloride ion concentration, with a maximum cooperativity at 150 mM NaCl. A mechanism is proposed that requires closure of the C-lobe before iron removal from the N-lobe can be effected; the open conformation of the C-lobe blocks a kinetically significant anion-binding site of the N-lobe, preventing its opening. Physiological implications of this cooperativity are discussed.     

Evolution reversed: the ability to bind iron restored to the N-lobe of the murine inhibitor of carbonic anhydrase by strategic mutagenesis

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.     

Structural reorganization of the transferrin C-lobe and transferrin receptor upon complex formation: the C-lobe binds to the receptor helical domain

Human transferrin, a bilobal protein, with each lobe bearing a single iron-binding site, functions to transport iron into cells. While the N-terminal lobe alone does not measurably bind cellular transferrin receptors or serve as an iron donor for cells, the C-lobe is capable of both functions. We used hydroxyl radical-mediated protein footprinting and mass spectrometry to reveal the conformational changes that occur upon complex formation for the human transferrin C-lobe (residues 334-679) bound to the ectodomain of human transferrin receptor 1 (residues 121-760). Oxidation rates for proteolytic peptides in the C-lobe, the receptor, and their complex have been measured by mass spectrometry; upon formation of the complex, a dramatic decrease in modification rates, indicating protection of specific side chain groups, can be seen in C-lobe sequences corresponding to residues 381-401, 415-433, and 457-470. Peptide sequences experiencing modification rate decreases in the transferrin receptor upon C-lobe binding include residues 232-240, 365-371, 496-508, 580 and 581, 614-623, 634-646, 647-681, and 733-760. In addition, several peptides in the receptor exhibit enhancements in the rate of modification consistent with allosteric effects of complex formation. Using tandem mass spectrometry, the sites of modification with altered reactivity in the complex include Met382, Met389, Trp460, Met464, and Phe427 in the C-lobe and Tyr503, Pro581, Tyr611, Leu619, Met635, Phe650, Trp740, Trp754, and Phe760 within the transferrin receptor. Using available genetic, biochemical, and structural data, we confirm that the conserved RGD sequence (residues 646-648) in the helical domain of the transferrin receptor, including residues from Leu619 to Phe650, is a primary binding site for the transferrin C-lobe.     

Iron release from transferrin, its C-lobe, and their complexes with transferrin receptor: presence of N-lobe accelerates release from C-lobe at endosomal pH

Human transferrin, like other members of the transferrin class of iron-binding proteins, is a bilobal structure, the product of duplication and fusion of an ancestral gene during the course of biochemical evolution. Although the two lobes exhibit 45% sequence identity and identical ligand structures of their iron-binding sites (one in each lobe), they differ in their iron-binding properties and their responsiveness to complex formation with the transferrin receptor. A variety of interlobe interactions modulating these iron-binding functions has been described. We have now studied the kinetics of iron release to pyrophosphate from the isolated recombinant C-lobe and from that lobe in the intact protein, each free and bound to receptor. The striking finding is that the rates of iron release at the pH of the endosome to which transferrin is internalized by the iron-dependent cell are similar in the free proteins but 18 times faster from full-length monoferric transferrin selectively loaded with iron in the C-lobe than from isolated C-lobe when each is complexed to the receptor. The possibility that the faster release in the receptor complex of the full-length protein at endosomal pH contributes to the evolutionary advantage of the bilobal structure is considered.     

Intrinsic fluorescence reports a global conformational change in the N-lobe of human serum transferrin following iron release

Transferrins have been extensively studied in order to understand how they reversibly bind and release iron. Human serum transferrin (hTF) is a single polypeptide chain that folds into two lobes (N- and C-lobe); each lobe binds a single ferric ion. Iron release induces a large conformational change in each lobe. At the putative endosomal pH of 5.6, measurement of the increase in intrinsic fluorescence upon iron release from the recombinant N-lobe yields two rate constants: 8.9 min-1 and 1.3 min-1. Direct monitoring of iron release from the N-lobe at pH 5.6 (by the decrease in absorbance at 470 nm) gives a single rate constant of 9.1 min-1, definitively establishing that the faster rate constant in the fluorescent studies is due to iron release. To further elucidate the molecular basis of the intrinsic fluorescence change (and the source of the slower rate constant), we examined the contributions of the three individual tryptophan residues in the N-lobe (Trp8, Trp128, and Trp264). Three double mutants, each containing the single remaining tryptophan residue, were produced. In the iron-bound N-lobe, Trp128 and Trp264 are quenched by iron and account for almost the entire fluorescent signal when iron is released. As for the wild-type N-lobe, the fluorescence increase for each of these mutants is best fit by a double-exponential function indicating two processes. Trp8 is severely quenched under all conditions, making virtually no contribution to the signal. Additionally, a mutant lacking all three Trp residues allows assignment of the fluorescent signal completely to the three tryptophan residues and observation of the presence of one (or more) tyrosinates in the N-lobe that have physiological significance in the uptake of iron.     

Expression,purification, and breast cancer cell inhibiting effect of recombinant human lactoferrin C-lobe

Lactoferrin (LTF), a multifunctional glycoprotein of the transferrin family mainly found in exotic secretions in mammals, is an important defense molecule against not only microbial invasion but also tumors. It folds into two globular domains (N- and C-lobes) each containing an iron-binding site. The cationic antimicrobial peptide in N-lobe is known to exert anti-tumor effect via a non-receptor-mediated pathway. However, whether LTF C-lobe also contributes to its anti-tumor activity remains to be investigated. In this study, a human LTF fragment (amino acid residues 343–682) covering the C-lobe was expressed with a histidine tag in E. coli and the purified polypeptide refolded through a series of buffer changing procedure. The resultant recombinant protein caused significant growth arrest of breast carcinoma cells MDA-MB-231 in a dose- and time-dependent manner, evidently via induction of apoptosis of the cell. Our data suggest a positive role for the C-lobe of human LTF in controlling tumors in vitro.     

Structural allostery and binding of the transferrin*receptor complex

The structural allostery and binding interface for the human serum transferrin (Tf)*transferrin receptor (TfR) complex were identified using radiolytic footprinting and mass spectrometry. We have determined previously that the transferrin C-lobe binds to the receptor helical domain. In this study we examined the binding interactions of full-length transferrin with receptor and compared these data with a model of the complex derived from cryoelectron microscopy (cryo-EM) reconstructions (Cheng, Y., Zak, O., Aisen, P., Harrison, S. C. & Walz, T. (2004) Structure of the human transferrin receptor.transferrin complex. Cell 116, 565-576). The footprinting results provide the following novel conclusions. First, we report characteristic oxidations of acidic residues in the C-lobe of native Tf and basic residues in the helical domain of TfR that were suppressed as a function of complex formation; this confirms ionic interactions between these protein segments as predicted by cryo-EM data and demonstrates a novel method for detecting ion pair interactions in the formation of macromolecular complexes. Second, the specific side-chain interactions between the C-lobe and N-lobe of transferrin and the corresponding interactions sites on the transferrin receptor predicted from cryo-EM were confirmed in solution. Last, the footprinting data revealed allosteric movements of the iron binding C- and N-lobes of Tf that sequester iron as a function of complex formation; these structural changes promote tighter binding of the metal ion and facilitate efficient ion transport during endocytosis.     

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.     

The Unique Kinetics of Iron Release from Transferrin: The Role of Receptor, Lobe-Lobe Interactions, and Salt at Endosomal pH

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 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 in the absence of the TFR, unequivocally establish 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-fold to 11-fold) and slows the rate of iron release from the N-lobe (6-fold 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 in the 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.     

The synergistic anion-binding sites of human transferrin: chemical and physiological effects of site-directed mutagenesis

A defining feature of all transferrins is the absolute dependence of iron binding on the concomitant binding of a synergistic anion, normally but not necessarily carbonate. Acting as a bridging ligand between iron and protein, it completes the coordination requirements of iron to lock the essential metal in its binding site. To investigate the role of the synergistic anion in the iron-binding and iron-donating properties of human transferrin, a bilobal protein with an iron binding site in each lobe, we have selectively mutated the anion-binding threonine and arginine ligands that form an essential part of the electrostatic and hydrogen-bonding network holding the synergistic anion to the protein. Preservation of either ligand is sufficient to maintain anion binding, and therefore iron binding, in the mutated lobe. Arginine is a stronger ligand than threonine, and its loss weakens carbonate and therefore iron binding, but maintains the ability of nitrilotriacetate to serve as a carbonate surrogate. Replacement of both ligands abolishes anion binding and consequently iron binding in the affected lobe. Loss of anion binding in either lobe results in a monoferric protein binding iron in normal fashion only in the opposite lobe. Both monoferric proteins are capable of transferrin receptor-dependent binding and iron donation to K562 cells, but with diminished receptor occupancy by the protein bearing iron only in the N-lobe.     

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).