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.     

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.     

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.     

A poly-His tag method for obtaining the C-terminal lobe of human transferrin

Human serum transferrin is an essential bilobal protein that transports iron in the circulation for delivery to iron-requiring cells. Obtaining the C-terminal lobe of human transferrin in verified native conformation has been problematic, possibly because its 11 disulfide bonds lead to misfolding when the lobe is expressed without its accompanying N-lobe. A recently reported method for preparing the C-lobe free of extraneous residues, with normal iron-binding properties and capable of delivering iron to cells, makes use of a Factor Xa cleavage site inserted into the interlobal connecting strand of the full-length protein. An inefficient step in this method requires the use of ConA chromatography to separate the cleaved lobes from each other, since only the C-lobe is glycosylated. Inserting a 6-His sequence near the start of the N-lobe enhances recovery of the recombinant transferrin from other proteins in the culture medium of the BHK21 cells expressing the mutant transferrin. The new procedure is more economical in time and effort than its predecessor, and offers the additional advantage of isolating C-lobe expressed with or without its glycan chains.     

Composition of pH-sensitive triad in C-lobe of human serum transferrin. Comparison to sequences of ovotransferrin and lactoferrin provides insight into functional differences in iron release

The transferrins (TF) are a family of bilobal glycoproteins that tightly bind ferric iron. Each of the homologous N- and C-lobes contains a single iron-binding site situated in a deep cleft. Human serum transferrin (hTF) serves as the iron transport protein in the blood; circulating transferrin binds to receptors on the cell surface, and the complex is internalized by endocytosis. Within the cell, a reduction in pH leads to iron release from hTF in a receptor-dependent process resulting in a large conformational change in each lobe. In the hTF N-lobe, two critical lysines facilitate this pH-dependent conformational change allowing entry of a chelator to capture the iron. In the C-lobe, the lysine pair is replaced by a triad of residues: Lys534, Arg632, and Asp634. Previous studies show that mutation of any of these triad residues to alanine results in significant retardation of iron release at both pH 7.4 and pH 5.6. In the present work, the role of the three residues is probed further by conversion to the residues observed at the equivalent positions in ovotransferrin (Q-K-L) and human lactoferrin (K-N-N) as well as a triad with an interchanged lysine and arginine (K534R/R632K). As expected, all of the constructs bind iron and associate with the receptor with nearly the same K(D) as the wild-type monoferric hTF control. However, interesting differences in the effect of the substitutions on the iron release rate in the presence and absence of the receptor at pH 5.6 are observed. Additionally, titration with KCl indicates that position 632 must have a positively charged residue to elicit a robust rate acceleration as a function of increasing salt. On the basis of these observations, a model for iron release from the hTF C-lobe is proposed. These studies provide insight into the importance of charge and geometry of the amino acids at these positions as a partial explanation for differences in behavior of individual TF family members, human serum transferrin, ovotransferrin, and lactoferrin. The studies collectively highlight important features common to both the N- and C-lobes of TF and the critical role of the receptor in iron release.     

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.     

Evidence that His349 acts as a pH-inducible switch to accelerate receptor-mediated iron release from the C-lobe of human transferrin

His349 in human transferrin (hTF) is a residue critical to transferrin receptor (TFR)-stimulated iron release from the C-lobe. To evaluate the importance of His349 on the TFR interaction, it was replaced by alanine, aspartate, lysine, leucine, tryptophan, and tyrosine in a monoferric C-lobe hTF construct (Fe_ChTF). Using a stopped-flow spectrofluorimeter, we determined rate processes assigned to iron release and conformational events (in the presence and in the absence of the TFR). Significantly, all mutant/TFR complexes feature dampened iron release rates. The critical contribution of His349 is most convincingly revealed by analysis of the kinetics as a function of pH (5.6–6.2). The Fe_ChTF/TFR complex titrates with a pK _a of approximately 5.9. By contrast, the H349A mutant/TFR complex releases iron at higher pH with a profile that is almost the inverse of that of the control complex. At the putative endosomal pH of 5.6 (in the presence of salt and chelator), iron is released from the H349W mutant/TFR and H349Y mutant/TFR complexes with a single rate constant similar to the iron release rate constant for the control; this suggests that these substitutions bypass the required pH-induced conformational change allowing the C-lobe to directly interact with the TFR to release iron. The H349K mutant proves that although the positive charge is crucial to complete iron release, the geometry at this position is also critical. The H349D mutant shows that a negative charge precludes complete iron release at pH 5.6 both in the presence and in the absence of the TFR. Thus, histidine uniquely drives the pH-induced conformational change in the C-lobe required for TFR interaction, which in turn promotes iron release.     

Receptor-modulated iron release from transferrin: differential effects on N- and C-terminal sites

Iron release to PPi from N- and C-terminal monoferric transferrins and their complexes with transferrin receptor has been studied at pH 7.4 and 5.6 in 0.05 M HEPES or MES/0.1 M NaCl/0.01 M CHAPS at 25 degrees C. The two sites exhibit kinetic heterogeneity in releasing iron. The N-terminal form is slightly less labile than its C-terminal counterpart at pH 7.4, but much more facile in releasing iron at pH 5.6. At pH 7.4, iron removal by 0.05 M pyrophosphate from each form of monoferric transferrin complexed to the receptor is considerably slower than from the corresponding free monoferric transferrin. However, at pH 5.6, complexation of transferrin to its receptor affects the two forms differently. The rate of iron release to 0.005 M pyrophosphate by the N-terminal species is substantially the same whether transferrin is free or bound to the receptor. In contrast, the C-terminal form releases iron much faster when complexed to the receptor than when free. Urea/PAGE analysis of iron removal from free and receptor-complexed diferric transferrin at pH 5.6 reveals that its C-terminal site is also more labile in the complex, but its N-terminal site is more labile in free diferric transferrin. Thus, the newly discovered role of transferrin receptor in modulating iron release from transferrin predominantly involves the C-terminal site. This observation helps explain the prevalence of circulating N-terminal monoferric transferrin in the human circulation.     

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.     

A new role for the transferrin receptor in the release of iron from transferrin

Iron removal by pyrophosphate from human serum diferric transferrin and the complex of transferrin with its receptor was studied in 0.05 M HEPES or MES buffers containing 0.1 M NaCl and 0.01 M CHAPS at 25 degrees C at pH 7.4, 6.4, and 5.6. At each pH, the concentration of pyrophosphate was adjusted to achieve rates of release amenable to study over a reasonable time course. Released iron was separated from protein-bound iron by poly(ethylene glycol) precipitation of aliquots drawn from the reaction mixture at various times during the course of a kinetic run. The amount of 59Fe label associated with the protein and pyrophosphate was determined from the radioactivity of precipitate and supernatant, respectively, in each aliquot. Iron removal of 0.05 M pyrophosphate at pH 7.4 from diferric transferrin bound to the receptor is considerably slower than that from free diferric transferrin, with observed pseudo-first-order rate constants of 0.020 and 0.191 min-1, respectively. For iron removal by 0.01 M pyrophosphate at pH 6.4, corresponding rate constants are 0.031 and 0.644 min-1. However, at pH 5.6, iron removal by 0.001 M pyrophosphate is faster from diferric transferrin bound to its receptor than from free transferrin (observed rate constants of 0.819 and 0.160 min-1, respectively). Thus, the transferrin receptor not only facilitates the removal of iron from diferric transferrin at the low pH that prevails in endocytic vesicles but may also reduce its accessibility to iron acceptors at extracellular pH, thereby minimizing the likelihood of nonspecific release of iron from transferrin at the cell surface.     

Receptor-induced switch in site-site cooperativity during iron release by transferrin.

Iron removal by PPi from the N- and C-terminal binding sites of both free and receptor-complexed transferrin, when the partner site remains occupied with kinetically inert Co(III), has been studied at pH 7.4 and 5.6, at 25 degrees C. At extracellular pH, 7.4, the C-terminal site of free mixed-metal proteins is slightly more labile than its N-terminal counterpart in releasing iron to 0.05 M PPi. The rate and extent of iron removal are retarded from both sites when transferrins are receptor-bound. At endosomal pH, 5.6, the two sites exhibit greater kinetic heterogeneity in iron release to 0.005 M PPi. The N-terminal site is 6 times more facile in relinquishing iron than the C-terminal site when mixed-metal transferrins are free. However, the two sites are affected oppositely upon binding to the receptor. Iron release from the C-terminal site of receptor-complexed CoN-transferrin-FeC is 4 times faster than that from receptor-free protein. In contrast, iron removal from the N-terminal site of receptor-complexed FeN-transferrin-CoC is slowed by a factor of 2 compared to that from free protein. These results help explain our previous observation of a receptor-induced switch in site lability during iron removal from diferric transferrin at pH 5.6 (Bali & Aisen, 1991). Site-site cooperative interactions between the two sites of doubly-occupied transferrin during iron release are altered upon binding to receptor at pH 5.6. Iron in the otherwise weaker binding site of the N-terminal lobe is stabilized, while iron in the relatively stable binding site of the C-terminal lobe is labilized.     

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.     

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

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

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.     

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

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

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.     

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.     

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.     

Studies on the mechanism of iron release from transferrin.

Iron release from human, rabbit, rat and sheep transferrin, chicken conalbumin and human lactoferrin was measured by the change in absorbance of solutions of the iron-protein complexes or by the release of 59Fe from the protein conjugated to agarose. Several phosphatic compounds and iron chelators were able to mediate the process (ATP, GTP, 2,3-diphosphoglycerate, inositol hexaphosphate, pyridoxal 5-phosphate, cytidine 5-triphosphate, pyrophosphate, inorganic phosphate, citrate, EDTA, oxalate, nitrilotriacetate). The greatest rate of iron release was found with pyrophosphate and the least with inorganic phosphate. Different rates of iron release were obtained with the different proteins, greatest with human transferrin and least with lactoferrin. With each of the proteins and the mediators there was a linera relationship between the H+ concentration and the rate of iron release. At any given pH the rate of iron release increased to a maximal rate as the mediator concentration was raised. It is concluded that iron release from transferrin under the conditions of these experiments involves an initial interaction between H+ and the iron-transferrin complex followed by release of the iron under the action of the mediator.     

Ligand variation in the transferrin family: the crystal structure of the H249Q mutant of the human transferrin N-lobe as a model for iron binding in insect transferrins

Proteins of the transferrin (Tf) family play a central role in iron homeostasis in vertebrates. In vertebrate Tfs, the four iron-binding ligands, 1 Asp, 2 Tyr, and 1 His, are invariant in both lobes of these bilobal proteins. In contrast, there are striking variations in the Tfs that have been characterized from insect species; in three of them, sequence changes in the C-lobe binding site render it nonfunctional, and in all of them the His ligand in the N-lobe site is changed to Gln. Surprisingly, mutagenesis of the histidine ligand, His249, to glutamine in the N-lobe half-molecule of human Tf (hTf/2N) shows that iron binding is destabilized and suggests that Gln249 does not bind to iron. We have determined the crystal structure of the H249Q mutant of hTf/2N and refined it at 1.85 A resolution (R = 0.221, R(free) = 0.246). The structure reveals that Gln249 does coordinate to iron, albeit with a lengthened Fe-Oepsilon1 bond of 2.34 A. In every other respect, the protein structure is unchanged from wild-type. Examination of insect Tf sequences shows that the K206.K296 dilysine pair, which aids iron release from the N-lobes of vertebrate Tfs, is not present in the insect proteins. We conclude that substitution of Gln for His does destabilize iron binding, but in the insect Tfs this is compensated by the loss of the dilysine interaction. The combination of a His ligand with the dilysine pair in vertebrate Tfs may have been a later evolutionary development that gives more sophisticated pH-mediated control of iron release from the N-lobe of transferrins.