Crystal structures and iron release properties of mutants (K206A and K296A) that abolish the dilysine interaction in the N-lobe of human transferrin

Human transferrin (Tf) is responsible for the binding and transport of iron in the bloodstream of vertebrates. Delivery of this bound iron to cells occurs by a process of receptor-mediated endocytosis during which Tf releases its iron at the reduced endosomal pH of approximately 5.6. Iron release from Tf involves a large conformational change in which the two domains that enclose the binding site in each lobe move apart. We have examined the role of two lysines, Lys206 and Lys296, that form a hydrogen-bonded pair close to the N-lobe binding site of human Tf and have been proposed to form a pH-sensitive trigger for iron release. We report high-resolution crystal structures for the K206A and K296A mutants of the N-lobe half-molecule of Tf, hTf/2N, and quantitative iron release data on these mutants and the double mutant K206A/K296A. The refined crystal structures (for K206A, R = 19.6% and R(free) = 23.7%; for K296A, R= 21.2% and R(free) = 29.5%) reveal a highly conserved hydrogen bonding network in the dilysine pair region that appears to be maintained even when individual hydrogen bonding groups change. The iron release data show that the mutants retain iron to a pH 1 unit lower than the pH limit of wild type hTf/2N, and release iron much more slowly as a result of the loss of the dilysine interaction. Added chloride ions are shown to accelerate iron release close to the pH at which iron is naturally lost and the closed structure becomes destabilized, and to retard it at higher pH.     

Crystal structure and iron-binding properties of the R210K mutant of the N-lobe of human lactoferrin: implications for iron release from transferrins

Lactoferrin (Lf) and serum transferrin (Tf) combine high-affinity iron binding with an ability to release this iron at reduced pH. Lf, however, retains iron to significantly lower pH than Tf, giving the two proteins distinct functional roles. In this paper, we compared the iron-release profiles for human Lf, Tf, and their N-lobe half-molecules Lf(N) and Tf(N) and showed that half of the difference in iron retention at low pH ( approximately 1.3 pH units) results from interlobe interactions in Lf. To probe factors intrinsic to the N-lobes, we further examined the specific role of two basic residues that are proposed to form a pH-sensitive dilysine trigger for iron release in the N-lobe of Tf [Dewan, J. C., Mikami, B., Hirose, M., and Sacchettini, J. C. (1993) Biochemistry 32, 11963-11968] by mutating Arg 210 to Lys in the N-lobe half-molecule Lf(N). The R210K mutant was expressed, purified, and crystallized, and its crystal structure was determined and refined at 2.0-A resolution to a final R factor (R(free)) of 19.8% (25.0%). The structure showed that Lys 210 and Lys 301 in R210K do not form a dilysine interaction like that between Lys 206 and Lys 296 in human Tf. The R210K mutant retained iron to lower pH than Tf(N), consistent with the absence of the dilysine interaction but released iron at approximately 0.7 pH units higher than Lf(N). We conclude that (i) the ability of Lf to retain iron to significantly lower pH than Tf is due equally to interlobe interactions and to the absence in Lfs of an interaction analogous to the dilysine pair in Tfs, even when two lysines are present at the corresponding sequence positions, and (ii) an appropriately positioned basic residue (Arg 210 in human Lf) modulates iron release by inhibiting protonation of the N-lobe iron ligands, specifically His 253.     

Mutation of the iron ligand His 249 to Glu in the N-lobe of human transferrin abolishes the dilysine "trigger" but does not significantly affect iron release

Serum transferrin is the major iron transport protein in humans. Its function depends on its ability to bind iron with very high affinity, yet to release this bound iron at the lower intracellular pH. Possible explanations for the release of iron from transferrin at low pH include protonation of a histidine ligand and the existence of a pH-sensitive "trigger" involving a hydrogen-bonded pair of lysines in the N-lobe of transferrin. We have determined the crystal structure of the His249Glu mutant of the N-lobe half-molecule of human transferrin and compared its iron-binding properties with those of the wild-type protein and other mutants. The crystal structure, determined at 2.4 A resolution (R-factor 19.8%, R(free) 29.4%), shows that Glu 249 is directly bound to iron, in place of the His ligand, and that a local movement of Lys 296 has broken the dilysine interaction. Despite the loss of this potentially pH-sensitive interaction, the H249E mutant is only slightly more acid-stable than wild-type and releases iron slightly faster. We conclude that the loss of the dilysine interaction does make the protein more acid stable but that this is counterbalanced by the replacement of a neutral ligand (His) by a negatively charged one (Glu), thus disrupting the electroneutrality of the binding site.     

Mutations at the histidine 249 ligand profoundly alter the spectral and iron-binding properties of human serum transferrin N-lobe

Human serum transferrin is an iron-binding and -transport protein which carries iron from the blood stream into various cells. Iron is held in two deep clefts located in the N- and C-lobes by coordinating to four amino acid ligands, Asp 63, Tyr 95, Tyr 188, and His 249 (N-lobe numbering), and to two oxygens from carbonate. We have previously reported the effect on the iron-binding properties of the N-lobe following mutation of the ligands Asp 63, Tyr 95, and Tyr 188. Here we report the profound functional changes which result from mutating His 249 to Ala, Glu, or Gln. The results are consistent with studies done in lactoferrin which showed that the histidine ligand is critical for the stability of the iron-binding site [H. Nicholson, B. F. Anderson, T. Bland, S. C. Shewry, J. W. Tweedie, and E. N. Baker (1997) Biochemistry 36, 341-346]. In the mutant H249A, the histidine ligand is disabled, resulting in a dramatic reduction in the kinetic stability of the protein toward loss of iron. The H249E mutant releases iron three times faster than wild-type protein but shows significant changes in both EPR spectra and the binding of anion. This appears to be the net effect of the metal ligand substitution from a neutral histidine residue to a negative glutamate residue and the disruption of the "dilysine trigger" [MacGillivray, R. T. A., Bewley, M. C., Smith, C. A., He, Q.-Y., Mason, A. B., Woodworth, R. C., and Baker, E. N. (2000) Biochemistry 39, 1211-1216]. In the H249Q mutant, Gln 249 appears not to directly contact the iron, given the similarity in the spectroscopic properties and the lability of iron release of this mutant to the H249A mutant. Further evidence for this idea is provided by the preference of both the H249A and H249Q mutants for nitrilotriacetate rather than carbonate in binding iron, probably because NTA is able to provide a third ligation partner. An intermediate species has been identified during the kinetic interconversion between the NTA and carbonate complexes of the H249A mutant. Thus, mutation of the His 249 residue does not abolish iron binding to the transferrin N-lobe but leads to the appearance of novel iron-binding sites of varying structure and stability.     

"Dilysine trigger" in transferrins probed by mutagenesis of lactoferrin: crystal structures of the R210G,R210E,and R210L mutants of human lactoferrin

The mammalian iron-binding proteins lactoferrin (Lf) and transferrin (Tf) bind iron very tightly, but reversibly. Despite homologous structures and essentially identical iron binding sites, Tf begins to release iron at pH 6.0, whereas Lf retains iron to pH approximately 3.5. This difference in iron retention gives the two proteins different biological roles. Two lysine residues, Lys 206 and Lys 296, which form a hydrogen-bonded dilysine pair in human Tf, have been shown to strongly influence iron release from the N-lobe. The equivalent residues in human Lf are Arg 210 and Lys 301, and we have here mutated Arg 210 in the N-lobe half-molecule of human lactoferrin, Lf(N), to probe its role in iron release. The Lf(N) mutants R210G, R210E, and R210L were expressed, purified, and crystallized, and their crystal structures were determined and refined at resolutions of 1.95 A (R210G), 2.2 A (R210E), and 2.0 A (R210L). The overall structures are very similar to that of wild-type Lf(N), but with small differences in domain orientations. In each of the mutants, however, Lys 301 (equivalent to Lys 296 in Tf) changes its conformation to fill the space occupied by Arg 210 Neta2 in wild-type Lf(N), interacting with the two tyrosine ligands Tyr 92 and Tyr 192. By comparison with other Lf and Tf structures, we conclude that Lys 301 (or Lys 296 in Tf) only occupies this site when residue 210 (206 in Tf) is nonpositive (neutral as in R210G and R210L or negative as in R210E). Thus, Lys 206 in the Tf dilysine pair is identified as having a depressed pK(a). Three specific sites are variably occupied by polar groups in the Lf mutants and other Lf and Tf proteins, and when coupled with iron-release data, these give new insights into the factors that most influence iron retention at low pH.     

Structural and functional studies on the stalk of the transferrin receptor

Transferrin (Tf) is an iron carrier protein that consists of two lobes, the N- and C-lobes, which can each bind a Fe³⁺ ion. Tf binds to its receptor (TfR), which mediates iron delivery to cells through an endocytotic pathway. Receptor binding facilitates iron release from the Tf C-lobe, but impedes iron release from the N-lobe. An atomic model of the Tf-TfR complex based on single particle electron microscopy (EM) indicated that receptor binding is indeed likely to hinder opening of the N-lobe, thus interfering with its iron release. The atomic model also suggested that the TfR stalks could form additional contacts with the Tf N-lobes, thus potentially further slowing down its iron release. Here, we show that the TfR stalks are unlikely to make strong interactions with the Tf N-lobes and that the stalks have no effect on iron release from the N-lobes of receptor-bound Tf.     

Effects of sialic acid residues of transferrin on the binding with aluminum and iron studied by HPLC/high-resolution ICP-MS

Transferrins (Tfs) are glycoproteins with carbohydrate chains in the C-lobe. Carbohydrate-deficient Tfs (CDTs) with fewer sialic acids increased in several diseases. In this study, the affinity of metals (Al and Fe) to Tfs was compared between native- and asialo-Tf by on-line high-performance liquid chromatography/high-resolution inductively coupled plasma mass spectrometry, to clarify whether the presence of sialic acids influences the metal binding. Fe added as Fe-citrate in the presence of bicarbonate preferred the N-lobe site and the binding affinity was similar between native- and asialo-Tfs. Al-citrate added at Al/Tf = 1 also preferred the N-lobe site, while the binding affinity was higher to asialo-Tf than to native-Tf. In Al-oxalate addition, the affinity to the N-lobe site of both Tfs increased further. In the absence of bicarbonate, Al-oxalate showed a preference for the C-lobe site in native-Tf and comparable affinity to both lobes in asialo-Tf. In asialo-Tf, Al2-Tf was the largest peak even at Al/Tf = 1. Thus, the lack of sialic acid in glycans and the presence of oxalate enhanced the binding affinity of Al to Tf. Therefore, it was suggested that the binding affinity of Al in patients with CDTs may be enhanced.     

Dual role of Lys206-Lys296 interaction in human transferrin N-lobe: iron-release trigger and anion-binding site.

The unique structural feature of the dilysine (Lys206-Lys296) pair in the transferrin N-lobe (hTF/2N) has been postulated to serve a special function in the release of iron from the protein. These two lysines, which are located in opposite domains, hydrogen bond to each other in the iron-containing hTF/2N at neutral pH but are far apart in the apo-form of the protein. It has been proposed that charge repulsion resulting from the protonation of the dilysines at lower pH may be the trigger to open the cleft and facilitate iron release. The fact that the dilysine pair is positively charged and resides in a location close to the metal-binding center has also led to the suggestion that the dilysine pair is an anion-binding site for chelators. The present report provides comprehensive evidence to confirm that the dilysine pair plays this dual role in modulating release of iron. When either of the lysines is mutated to glutamate or glutamine or when both are mutated to glutamate, release of iron is much slower compared to the wild-type protein. This is due to the fact that the driving force for cleft opening is absent in the mutants or is converted to a lock-like interaction (in the case of the K206E and K296E mutants). Direct titration of the apo-proteins with anions as well as anion-dependent iron release studies show that the dilysine pair is part of an active anion-binding site which exists with the Lys296-Tyr188 interaction as a core. At this site, Lys296 serves as the primary anion-binding residue and Tyr188 is the main reporter for electronic spectral change, with smaller contributions from Lys206, Tyr85, and Tyr95. In iron-loaded hTF/2N, anion binding becomes invisible as monitored by UV-vis difference spectra since the spectral reporters Tyr188 and Tyr95 are bound to iron. Our data strongly support the hypothesis that the apo-hTF/2N exists in equilibrium between the open and closed conformations, because only in the closed form is Lys296 in direct contact with Tyr188. The current findings bring together observations, ideas, and experimental data from a large number of previous studies and shed further light on the detailed mechanism of iron release from the transferrin N-lobe. In iron-containing hTF/2N, Lys296 may still function as a target to introduce an anion (or a chelator) near to the iron-binding center. When the pH is lowered, the protonation of carbonate (synergistic anion for metal binding) and then the dilysine pair form the driving force to loosen the cleft, exposing iron; the nearby anion (or chelator) then binds to the iron and releases it from the protein.     

Structural and functional consequences of binding site mutations in transferrin: crystal structures of the Asp63Glu and Arg124Ala mutants of the N-lobe of human transferrin

Human transferrin is a serum protein whose function is to bind Fe(3+) with very high affinity and transport it to cells, for delivery by receptor-mediated endocytosis. Structurally, the transferrin molecule is folded into two globular lobes, representing its N-terminal and C-terminal halves, with each lobe possessing a high-affinity iron binding site, in a cleft between two domains. Central to function is a highly conserved set of iron ligands, including an aspartate residue (Asp63 in the N-lobe) that also hydrogen bonds between the two domains and an arginine residue (Arg124 in the N-lobe) that binds an iron-bound carbonate ion. To further probe the roles of these residues, we have determined the crystal structures of the D63E and R124A mutants of the N-terminal half-molecule of human transferrin. The structure of the D63E mutant, determined at 1.9 A resolution (R = 0.245, R(free) = 0.261), showed that the carboxyl group still binds to iron despite the larger size of the Glu side chain, with some slight rearrangement of the first turn of alpha-helix residues 63-72, to which it is attached. The structure of the R124A mutant, determined at 2.4 A resolution (R = 0.219, R(free) = 0.288), shows that the loss of the arginine side chain results in a 0.3 A displacement of the carbonate ion, and an accompanying movement of the iron atom. In both mutants, the iron coordination is changed slightly, the principal change being in each case a lengthening of the Fe-N(His249) bond. Both mutants also release iron more readily than the wild type, kinetically and in terms of acid lability of iron binding. We attribute this to more facile protonation of the synergistically bound carbonate ion, in the case of R124A, and to strain resulting from the accommodation of the larger Glu side chain, in the case of D63E. In both cases, the weakened Fe-N(His) bond may also contribute, consistent with protonation of the His ligand being an early intermediate step in iron release, following the protonation of the carbonate ion.     

Titanium preferential binding sites in human serum transferrin at physiological concentrations

Serum transferrin (Tf) is an iron binding glycoprotein that plays a central role in the metabolism of this essential metal but it also binds other metal ions. Four main transferrin forms containing different iron binding states can be distinguished in human serum samples: monoferric (C-site or N-site), holotransferrin (with two Fe atoms) and apotransferrin (with no metal). Recently, it has been reported that Tf binds also Ti even more tightly than does Fe, in artificially Ti(iv) spiked solutions. However, very limited work has been done on the Ti binding to Tf at physiological concentrations in patients carrying intramedullary Ti nails. Here we report the chemical association of Ti to Tf "in vivo" under different chromatographic conditions by elemental mass spectrometry using double focusing inductively coupled plasma (DF-ICP-MS) as detector. For the separation of the Ti/Fe-Tf forms different gradient conditions have been explored. The observed results reveal that human serum Ti (from patients carrying intramedullary Ti nails) is uniquely associated to the N-lobe of Tf. The investigation of the influence of sialic acid in the carbohydrate chain of human serum Tf, studied by incubating the protein with neuraminidase (sialidase) to obtain the monosialilated species, revealed that the binding affinity of Ti was similar for monosialo-Tf and for native-Tf and occurs in the N-lobe. These results suggest that the species Fe(C)Ti(N)-TF might provide a route for Ti entry into cells via the transferrin receptors after the release of the metal from its implants.     

Production of transferrin receptors by Histophilus ovis: three of five strains require two signals

Five strains of Histophilus ovis (9L, 642A, 714, 5688T, and 3384Y) were investigated with respect to iron acquisition. All strains used ovine, bovine, and goat transferrins (Tfs), but not porcine or human Tfs, as iron sources for growth. In solid phase binding assays, total membranes from only two (9L and 642A) of the five strains, grown under iron-restricted conditions, were able to bind Tfs (ovine, bovine, and goat, but not porcine or human). However, when the organisms were grown under iron-restricted conditions in the presence of bovine transferrin (Tf), total membranes from all strains exhibited Tf binding (as above); competition experiments demonstrated that all three Tfs (ovine, bovine, and goat) were bound by the same receptor(s). Membranes from organisms grown under iron-replete conditions in the presence or absence of bovine Tf failed to bind any of the test Tfs. An affinity-isolation procedure allowed the isolation of two putative Tf-binding polypeptides (78 and 66 kDa) from total membranes of strains 9L and 642A grown under iron-restricted conditions, and from membranes of all strains if the growth medium also contained Tf. It is concluded that all strains tested acquire Tf-bound iron by means of siderophore-independent mechanisms involving surface receptors analogous to the Tf-binding proteins (TbpA and TbpB) found in comparable organisms; although iron restriction alone is sufficient to promote the expression of these proteins by strains 9L and 642A, their production by strains 714, 5688T, and 3384Y appears to require two signals, iron restriction and the presence of Tf.     

A simple method for obtaining transferrins from human plasma and porcine serum: preparations and properties

A simple method was described for the purification of serum transferrin (Tf) from human plasma and porcine serum with relative high yield and purity. The properties including purity, integrity, immunoreactivity and the receptor-binding ability of the proteins were studied by several assays, comprising spectrometry, SDS-PAGE, HPLC, Western blotting, urea electrophoresis, mass spectrometry and cytometry. Analysis from all the different aspects manifested that the proteins were of high purity. The two kinds of Tfs appeared to be iron-saturated as confirmed by their absorbance spectra and urea-PAGE mobility. The specific spectra of absorption of the two Tfs were both at around 465 nm. The relative molecular weights of human Tf (hTf) and porcine Tf (pTf) were determined by SDS-PAGE and further identified by MAIDI-TOF mass spectrometry with a result of 79,707 and 79,258, respectively. Immunoblotting assay showed that pTf could react with the anti-human Tf monoclonal antibody with a less level compared to hTf. FACS assays of their binding activities to Tf receptor-positive cell (K562 cell line) indicated that pTf could be recognized by the hTf receptor and internalized into cells, with a slightly less efficacy than hTf. All special property studies demonstrated that pTf was similar to hTf in physical and chemical characteristics, which gave a hint that pTf could substitute for hTf in some kinds of researches, such as using hTf as a carrier in drug targeting system.     

Recombinant human transferrin: beyond iron binding and transport

Iron is indispensible for life and essential for such processes as oxygen transport, electron transfer and DNA synthesis. Transferrin (Tf) is a ubiquitous protein with a central role in iron transport and metabolism. There is evidence, however, that Tf has many other biological roles in addition to its primary function of facilitating iron transport and metabolism, such as its profound effect on mammalian cell growth and productivity. The multiple functions of Tf can be exploited to develop many novel applications. Indeed, over the past several years, considerable efforts have been directed towards exploring human serum Tf (hTf), especially the use of recombinant native hTf and recombinant Tf fusion proteins, for various applications within biotechnology and medicine. Here, we review some of the remarkable progress that has been made towards the application of hTf in these diverse areas and discuss some of the exciting future prospects for hTf.     

Binding patterns of vanadium ions with different valence states to human serum transferrin studied by HPLC/high-resolution ICP-MS

Vanadium (V) is an essential metal for mammals and has different valence states. In blood, V is bound to serum transferrin (Tf), a glycoprotein which has two metal-binding sites, and carbonate is generally required for the binding. In this study, the binding patterns of V(III), V(IV), and V(V) to human serum Tf (hTf) were analyzed using an HPLC system equipped with an anion-exchange column and directly connected to a high-resolution inductively coupled plasma-mass spectrometer for metal detection (51V). In affinity to hTf, the three ions were ranked V(III)>V(IV)>V(V) in the presence of bicarbonate and V(III) reverse congruent V(IV)>V(V) in the absence. Intermediates in the "open forms" binding to the respective sites were detected at the initial stage. V(IV) and V(V) were bound to the N-lobe site in the "closed form" and "open form," respectively. In the absence of bicarbonate, V ions with respective valence states were bound to hTf in the "open form." In terms of binding to hTf, tri-valent V was most favorable in the presence of bicarbonate.     

Differed preferential iron-binding lobe in human transferrin depending on the presence of bicarbonate detected by HPLC/high-resolution inductively coupled plasma mass spectrometry

The binding of iron (Fe) to human serum transferrin (Tf) was analyzed with an HPLC system equipped with an anion exchange column and directly connected with a high-resolution inductively coupled plasma mass spectrometer for metal detection. The (56)Fe level in the eluate was monitored at resolution m/Deltam=3000. Two monoferric Tfs were assigned based on the results of urea-PAGE and desferrioxamine experiments. When Fe was added as Fe-citrate stepwise to an apo-Tf solution in the presence of bicarbonate, the N-lobe site was the preferential Fe-binding site, while the C-lobe site was preferred in the absence of bicarbonate. In both cases, the Fe-peak areas of the preferential site and Fe(2)-Tf increased up to an Fe/Tf molar ratio of 1, and then the peak area of the monoferric Tf decreased while the peak area of Fe(2)-Tf increased. When the Fe/Tf molar ratio was below 1, the amount of Fe bound to the lobe with a weaker affinity was higher in Fe(2)-Tf than in the monoferric Tf in each case. Namely, Fe(2)-Tf was the preferential binding state of Fe to human serum Tf. The preference is reasonable for transferring Fe ions effectively to Tf-receptors.     

Class specificity of transferrin as a muscle trophic factor

The specificity of transferrin (Tf) in its exertion of a growth-promoting effect on myogenic cells was examined using serum Tfs from chick, dove, goose, turkey, bovine, horse, rabbit, rat, and swine and primary myogenic cells from chick, duck, quail, rabbit, and rat, and rat L6 cells. Avian Tfs were effective on avian cells but not on mammalian cells, while mammalian Tfs were effective on mammalian cells but not on avian cells. Dove and bovine Tfs were exceptional in that they were effective on some class-heterologous cells at higher concentrations and less so or completely ineffective on some class-homologous cells. Despite these exceptions, however, the relationship between Tfs and cells can be summarized as a class specificity. To exert the growth-promoting effect, it is prerequisite for Tf to bind its specific receptor on the cell surface. Using quail and L6 cells, we found that the binding of 125I-labeled chick and rat Tfs to the respective receptors of quail and L6 myoblasts was competitively inhibited by other kinds of effective Tfs, but not by ineffective ones. We conclude that the class specificity in myotrophic activity of Tf is due to the affinity between Tf and Tf receptor.     

Binding affinity of aluminium to human serum transferrin and effects of carbohydrate chain modification as studied by HPLC/high-resolution ICP-MS--speciation of aluminium in human serum

Aluminium (Al) in the blood is bound to transferrin (Tf), a glycoprotein of about 80kDa that is characterized by its need for a synergistic anion. In this focused review, the binding affinity of Al to Tf is surveyed in the context of our recent studies using on-line high-performance liquid chromatography/high-resolution inductively coupled plasma mass spectrometry (HPLC/HR-ICP-MS). Al in human serum without any in vitro Al-spikes was present in a form bound to the N-lobe site of Tf. The influences of sialic acid in the carbohydrate chain of human serum Tf (hTf) were studied using asialo-hTf, obtained by treatment with sialidase. The binding affinity of Fe was similar between asialo-hTf and native-hTf, while that of Al for asialo-hTf was larger than that for native-hTf, especially in the presence of oxalate, a synergistic anion. The above findings are discussed in relation to diseases in which the serum concentrations of carbohydrate-deficient Tf and oxalate are augmented.     

Mechanism for multiple ligand recognition by the human transferrin receptor

Transferrin receptor 1 (TfR) plays a critical role in cellular iron import for most higher organisms. Cell surface TfR binds to circulating iron-loaded transferrin (Fe-Tf) and transports it to acidic endosomes, where low pH promotes iron to dissociate from transferrin (Tf) in a TfR-assisted process. The iron-free form of Tf (apo-Tf) remains bound to TfR and is recycled to the cell surface, where the complex dissociates upon exposure to the slightly basic pH of the blood. Fe-Tf competes for binding to TfR with HFE, the protein mutated in the iron-overload disease hereditary hemochromatosis. We used a quantitative surface plasmon resonance assay to determine the binding affinities of an extensive set of site-directed TfR mutants to HFE and Fe-Tf at pH 7.4 and to apo-Tf at pH 6.3. These results confirm the previous finding that Fe-Tf and HFE compete for the receptor by binding to an overlapping site on the TfR helical domain. Spatially distant mutations in the TfR protease-like domain affect binding of Fe-Tf, but not iron-loaded Tf C-lobe, apo-Tf, or HFE, and mutations at the edge of the TfR helical domain affect binding of apo-Tf, but not Fe-Tf or HFE. The binding data presented here reveal the binding footprints on TfR for Fe-Tf and apo-Tf. These data support a model in which the Tf C-lobe contacts the TfR helical domain and the Tf N-lobe contacts the base of the TfR protease-like domain. The differential effects of some TfR mutations on binding to Fe-Tf and apo-Tf suggest differences in the contact points between TfR and the two forms of Tf that could be caused by pH-dependent conformational changes in Tf, TfR, or both. From these data, we propose a structure-based model for the mechanism of TfR-assisted iron release from Fe-Tf.     

Polymorphic variation in the amount of sialic acid attached to bovine transferrin

In this paper family studies are presented which support the hypothesis of polymorphism in the process controlling sialic acid binding to bovine transferrin which modifies its phenotype as seen in starch gel electrophoresis. It has been shown that this polymorphism is controlled by a locus Tfs with two alleles Tfs A and Tfs a. Tfs a/a animals have the abnormal phenotype with the two faster bands of the four bands of a normal transferrin allele being virtually absent. Tfs A/a and Tfs A/A are phenotypically normal. Limited evidence is presented which suggests that the Tf and Tfs loci are not linked.     

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.