Nonequivalence of the metal binding sites in vanadyl-labeled human serum transferrin.

Vanadyl ion, VO(IV), has been used as an electron paramagnetic resonance (EPR) spin label to study the metal-binding properties of human serum transferrin in the presence of bicarbonate. Iron-saturated transferrin does not bind the vanadyl ion. Room temperature titrations of apotransferrin with VO(IV) as monitored by EPR indicate the extent of binding to be pH dependent, with a full 0.2 VO(IV) ions per transferrin molecule bound at pH 7.5 and 9, but only about 1.2 VO(IV) ions bound at pH 6. The EPR spectra of frozen solutions with or without 0.1 M NaCUO4 at 77 K show that there are two spectroscopically nonequivalent binding sites (A and B) with a slight difference in binding constants. One site (A site) exhibits essentially constant binding capacity in the pH range 6-9, but the other (B site) becomes less avialable as the pH is reduced below 7. Results with mixed Fe(III)-VO(IV) transferrin complexes suggest that iron shows a slight tendency to bind at the B site over the A site pH 7.5 and 9.0. Only the B site in both vanadyl and iron transferrins is perturbed by the presence of perchlorate.     

Thermodynamics of anion binding to human serum transferrin

The binding of phosphate, bicarbonate, sulfate, and vanadate to human serum transferrin has been evaluated by two difference ultraviolet spectroscopic techniques. Direct titration of apotransferrin with bicarbonate, phosphate, and sulfate produces a strong negative absorbance near 245 nm, while titration with vanadate produces a positive absorbance in this region. Least-squares refinement of the absorbance data indicates that two anions of sulfate, phosphate, and vanadate bind to each transferrin molecule but that there is detectable binding of only a single bicarbonate anion. A second method used to study the thermodynamics of anion binding was competition equilibrium between anions for binding to the transferrin. The equilibrium constant for binding of the first equivalent of vanadate was determined by competition vs. phosphate and sulfate, while the equilibrium constant for binding of the second equivalent of bicarbonate was determined by competition vs. vanadate. Anion binding was described by two equilibrium constants for the successive binding of two anions per transferrin molecule: K1 = [A-Tr]/[A][Tr] and K2 = [A-Tr-A]/[A][A-Tr] where [A] represents the free anion concentration, [Tr] represents apotransferrin concentration, and [A-Tr] and [A-Tr-A] represent the concentrations of 1:1 and 2:1 anion-transferrin complexes, respectively. The results were the following: for phosphate, log K1 = 4.19 +/- 0.03 and log K2 = 3.25 +/- 0.21; for sulfate, log K1 = 3.62 +/- 0.07 and log K2 = 2.79 +/- 0.20; for vanadate, log K1 = 7.45 +/- 0.10 and log K2 = 6.6 +/- 0.30; for bicarbonate, log K1 = 2.66 +/- 0.07 and log K2 = 1.8 +/- 0.3.(ABSTRACT TRUNCATED AT 250 WORDS)     

Steric and allosteric factors prevent simultaneous binding of transferrin-binding proteins A and B to transferrin

The ability to acquire iron directly from host Tf (transferrin) is an adaptation common to important bacterial pathogens belonging to the Pasteurellaceae, Moraxellaceae and Neisseriaceae families. A surface receptor comprising an integral outer membrane protein, TbpA (Tf-binding protein A), and a surface-exposed lipoprotein, TbpB (Tf-binding protein B), mediates the iron acquisition process. TbpB is thought to extend from the cell surface for capture of Tf to initiate the process and deliver Tf to TbpA. TbpA functions as a gated channel for the passage of iron into the periplasm. In the present study we have mapped the effect of TbpA from Actinobacillus pleuropneumoniae on pTf (porcine Tf) using H/DX-MS (hydrogen/deuterium exchange coupled to MS) and compare it with a previously determined binding site for TbpB. The proposed TbpA footprint is adjacent to and potentially overlapping the TbpB-binding site, and induces a structural instability in the TbpB site. This suggests that simultaneous binding to pTf by both receptors would be hindered. We demonstrate that a recombinant TbpB lacking a portion of its anchor peptide is unable to form a stable ternary TbpA-pTf-TbpB complex. This truncated TbpB does not bind to a preformed Tf-TbpA complex, and TbpA removes pTf from a preformed Tf-TbpB complex. Thus the results of the present study support a model whereby TbpB 'hands-off' pTf to TbpA, which completes the iron removal and transport process.     

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.     

Site selectivity in the binding of inorganic anions to serum transferrin

Equilibrium constants for the sequential binding of two anions at the specific metal-binding sites of apotransferrin have been measured by difference ultraviolet spectroscopy in 0.1 M N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (Hepes) at pH 7.4 and 25 degrees C. Log K1 values for phosphate, phosphite, sulfate, and arsenate fall in the narrow range of 3.5-4.0, while the log K1 for bicarbonate is 2.73. No binding is observed for nitrate, perchlorate, or borate. A dinegative charge appears to be the most important criterion for anion binding. Equilibrium constants have also been measured for binding of anions to both forms of mono(ferric)transferrin. There appears to be a very small site selectivity (0.2 to 0.4 log units) for phosphate, arsenate, and phosphite that favors binding to the N-terminal site, but there is no detectable selectivity for binding of sulfate or bicarbonate. Comparison of the binding affinities and anion selectivity with literature data on anion-binding to protonated macrocyles and cryptates strongly supports the existence of specific anion-binding sites on the protein. Binding constants were also measured in 0.01 M Hepes. The anionic sulfonate group of the buffer appears to have a small effect on anion binding.     

Effect of metal ions on binding of bilirubin to erythrocyte membranes

Binding of bilirubin to different erythrocyte membranes, namely, human, buffalo, sheep and goat, pre-incubated with different concentrations of metal ions was studied. The results showed that among the different metal ions used, Ca2+ had the highest potential in increasing the amount of bound bilirubin followed by Sr2+ and Mg2+, whereas Ba2+ had the lowest potential. Treatment of these membranes with Ca2+ led to an increase in the amount of bound bilirubin in all membranes. However, human erythrocyte membranes pretreated with Ca2+, bound the highest amount of bilirubin compared to other erythrocyte membranes. Increase in bilirubin binding upon Ca2+-treatment can be ascribed to shielding effect, redistribution of phospholipids as well as increase in surface hydrophobicity induced by Ca2+.     

Equilibrium studies on the binding of cadmium(II) to human serum transferrin

The binding of cadmium(II) to human serum transferrin in 0.01 M N-(2-hydroxyethyl)-piperazine-N'-2-ethanesulfonic acid with 5 mM bicarbonate at 25 degrees C has been evaluated by difference ultraviolet spectroscopy. Equilibrium constants were determined by competition versus three different low molecular weight chelating agents: nitrilotriacetic acid, ethylenediamine-N,N'-diacetic acid, and triethylenetetramine. Conditional equilibrium constants for the sequential binding of two cadmium ions to transferrin under the stated experimental conditions are log K1 = 5.95 +/- 0.10 and log K2 = 4.86 +/- 0.13. A linear free energy relationship for the complexation of cadmium and zinc has been prepared by using equilibrium data on 243 complexes of these metal ions with low molecular weight ligands. The transferrin binding constants for cadmium and zinc are in good agreement with this linear free energy relationship. This indicates that the larger size of the cadmium(II) ion does not significantly hinder its binding to the protein.     

The binding of metal ions to bovine factor IX

The binding isotherms of Ca2+ and Mn2+ to bovine factor IX have been determined at pH 6.5 and pH 7.4, at 25 degrees C. At pH 7.4, at least 2 strong Ca2+ sites, with an average KDISS of 0.1 +/- 0.02 mM, are found. An additional 10 to 11 weaker Ca2+ binding sites, with an average KDISS of 1.3 +/- 0.2 mM are noted, at high levels of Ca2+. At pH 6.5, again at least 2 strong Ca2+ sites on factor IX are evident, with an average KDISS of 0.11 +/- 0.02 mM; but slightly fewer (7 to 8) weaker sites, with an average KDISS of 1.9 +/- 0.3 mM, are obtained. Qualitatively, the binding of Mn2+ to bovine factor IX appears similar to that of Ca2+. At pH 6.5, approximately 2 strong Mn2+ binding sites, with an average KDISS of 13 +/- 1.5 micrometer, and an additional 5 to 6 weak sites, with an average KDISS of 160 +/- 15 micrometer, are present. Manganese does not completely displace Ca2+; and Ca2+ does not completely displace Mn2+ from their respective binding sites. On the other hand, Tb3+ and Sm3+ readily displace Ca2+, at pH 6.5, from its sites on factor IX. The rate and extent of activation of bovine factor IX, by bovine factor XIa, is dependent on the Ca2+ concentration, up to concentrations of Ca2+ which saturate its effect on the system. Substitution of Sr2+ for Ca2+ leads to approximately 50% of the maximum rate of factor IXa formation, and final yield of factor IXa, in this activation system. Manganese does not substitute for Ca2+ in this activation, but does inhibit the stimulatory effect of Ca2+. Both Tb3+ and Sm3+ are effective inhibitors of Ca2+ in factor IX activation by factor XIa.     

In vitro study on the binding of anti-coagulant vitamin to bovine serum albumin and the influence of toxic ions and common ions on binding

The mechanism of binding of vitamin K(3) (VK(3)) with bovine serum albumin (BSA) was investigated by fluorescence, absorption and circular dichroism (CD) techniques under physiological conditions. The analysis of fluorescence data indicated the presence of static quenching mechanism in the binding. Various binding parameters have been evaluated. Thermodynamic parameters, the standard enthalpy change, DeltaH(0) and the standard entropy change, DeltaS(0) were observed to be -164.09 kJ mol(-1) and -465.08 J mol(-1)K, respectively. The quantitative analysis of CD spectra confirmed the change in secondary structure of the protein upon interaction with VK(3). The binding average distance, r between the donor (BSA) and acceptor (VK(3)) was determined based on the F?rster's theory and it was found to be 3.3 nm. The effects of toxic ions and common ions on VK(3)-BSA system were also investigated.     

Glycan chains modulate prion protein binding to immobilized metal ions

PrP(c) is the normal isoform of the prion protein which can be converted into PrP(Sc), the pathology-associated conformer in prion diseases. It contains two N-linked glycan chains attached to the C-proximal globular domain. While the biological functions of PrP(c) are still unknown, its ability to bind Cu(2+) is well documented. The main Cu(2+)-binding sites are located in the N-proximal, unstructured region of the molecule. Here we report that PrP(c) glycans influence the capacity of PrP(c) from sheep brain or cultured Rov cells to bind IMAC columns loaded with Cu(2+) or Co(2+). Using different anti-PrP antibodies and PrP(c) glycosylation mutants, we show that the full length non-glycosylated form of PrP(c) has a higher binding efficiency for column-bound Cu(2+) and Co(2+) than the corresponding glycosylated form. Our findings raise the possibility that the accessibility of the PrP(c) metal ion-binding sites might be controlled by the glycan chains.     

Unusual features for zirconium(IV) binding to human serum transferrin

Human serum apotransferrin (hTF) binds to Zr(IV) slowly in the presence of nitrilotriacetate (NTA), citrate or ethylenediaminetetraacetate (EDTA) as donor ligands. For Zr(NTA)(2)(2-) as donor, equilibrium was reached in ca. 2 h (pH 7.4, 298 K, 10 mM Hepes, 5 mM bicarbonate) and full loading of the N- and C-lobe sites was achievable to give Zr(2)-hTF. (13)C NMR data suggest that carbonate can bind as a synergistic anion. (1)H and 2D [(1)H,(13)C] (using epsilon-[(13)C]Met-hTF) NMR studies show that there is little lobe-selectively in the order of Zr(IV) uptake. Fe(III) displaced Zr(IV) from the C-lobe of Zr(2)-hTF first, followed by the N-lobe. However, in the presence of a large excess of NTA, Zr(IV) binds to the N-lobe of holo-hTF (Fe(2)-hTF) first followed by the C-lobe. The (1)H and (13)C NMR chemical shift changes for epsilon-[(13)CH(3)] of Met464, which is close to the C-lobe site, are quite distinct from those observed previously for Al(III), Fe(III), Ti(IV), Ga(III) and Bi(III) binding to hTF, suggesting that Zr(IV) binding may not induce lobe closure [as observed previously for Hf(IV)]. This may affect receptor recognition and play a role in the different biological behaviour of Zr(IV) compared to Ti(IV).     

Iodination significantly influences the binding of human transferrin to the transferrin receptor

The human transferrin receptor (TfR) and its ligand, the serum iron carrier transferrin, serve as a model system for endocytic receptors. Although the complete structure of the receptor's ectodomain and a partial structure of the ligand have been published, conflicting results still exist about the magnitude of equilibrium binding constants, possibly due to different labeling techniques. In the present study, we determined the equilibrium binding constant of purified human TfR and transferrin. The results were compared to those obtained with either iodinated TfR or transferrin. Using an enzyme-linked assay for receptor-ligand interactions based on the published direct calibration ELISA technique, we determined an equilibrium constant of Kd=0.22 nM for the binding of unmodified human Tf to surface-immobilized human TfR. In a reciprocal experiment using soluble receptor and surface-bound transferrin, a similar constant of Kd=0.23 nM was measured. In contrast, covalent labeling of either TfR or transferrin with 125I reduced the affinity 3-5-fold to Kd=0.66 nM and Kd=1.01 nM, respectively. The decrease in affinity upon iodination of transferrin is contrasted by an only 1.9-fold decrease in the association rate constant, suggesting that the iodination affects rather the dissociation than the association kinetics. These results indicate that precautions should be taken when interpreting equilibrium and rate constants determined with covalently labeled components.     

Binding of transferrin and metal ions by suspensions of reticulocyte-rich rabbit blood

Reticulocyte binding of Fe(III)_-transferrin and transferrin complexes with other metal ions have been compared by different investigators. The functional relevance of this comparison is not clear, therefore transferrin complexes with Fe(III), Cu(II), Mn(II) and Zn(II) have been studied further by DEAE-cellulose chromatography and by measurement of transferrin and metal uptakes by rabbit reticulocytes.Human Fe-transferrin behaved as a weaker anion than apotransferrin during DEAE-cellulose chromatography; since Fe-transferrin has a higher negative charge than apotransferrin and behaves a as stronger anion in electrophoretic systems, the chromatographic result was the opposite of that anticipated. The lower affinity of human Fe-transferrin for DEAE-cellulose is probably caused by a redistribution of charged groups on the surface of transferrin molecules when Fe(III) ions are bound and is therefore considered to be dependent on molecular conformation. Apotransferrin and divalent metal-transferrin complexes were found to have nearly equal affinities for DEAE-cellulose, thus the effect on surface charge of human transferrin molecules induced by binding Fe(III) appeared to be limited to that metal ion.Iron uptake by reticulocytes was associated with increased binding of transferrin to the cell surface: uptake of divalent metals occured without a concomitant increase in transferrin uptake or evidence of a specific metal-transfer process. Cu-transferrin was rapidly dissociated during incubation with cells.The effect of Fe(III)_binding on human transferrin molecules was to alter the molecular affinity for charged surfaces, namely DEAE-cellulose and reticulocyte membranes. This was less apparent with rabbit transferrin. Transferrin complexes with divalent metals behaved as apotransferrin in the process of association with reticulocytes.     

The contribution of metal ions to the structural stability of the large ribosomal subunit

Both monovalent cations and magnesium ions are well known to be essential for the folding and stability of large RNA molecules that form complex and compact structures. In the atomic structure of the large ribosomal subunit from Haloarcula marismortui, we have identified 116 magnesium ions and 88 monovalent cations bound principally to rRNA. Although the rRNA structures to which these metal ions bind are highly idiosyncratic, a few common principles have emerged from the identities of the specific functional groups that coordinate them. The nonbridging oxygen of a phosphate group is the most common inner shell ligand of Mg++, and Mg++ ions having one or two such inner shell ligands are very common. Nonbridging phosphate oxygens and the heteroatoms of nucleotide bases are common outer shell ligands for Mg++ ions. Monovalent cations usually interact with nucleotide bases and protein groups, although some interactions with nonbridging phosphate oxygens are found. The most common monovalent cation binding site is the major groove side of G-U wobble pairs. Both divalent and monovalent cations stabilize the tertiary structure of 23S rRNA by mediating interactions between its structural domains. Bound metal ions are particularly abundant in the region surrounding the peptidyl transferase center, where stabilizing cationic tails of ribosomal proteins are notably absent. This may point to the importance of metal ions for the stabilization of specific RNA structures in the evolutionary period prior to the appearance of proteins, and hence many of these metal ion binding sites may be conserved across all phylogenetic kingdoms.     

Dissecting ITC data of metal ions binding to ligands and proteins

BackgroundITC is a powerful technique that can reliably assess the thermodynamic underpinnings of a wide range of binding events. When metal ions are involved, complications arise in evaluating the data due to unavoidable solution chemistry that includes metal speciation and a variety of linked equilibria.Scope of reviewThis paper identifies these concerns, provides recommendations to avoid common mistakes, and guides the reader through the mathematical treatment of ITC data to arrive at a set of thermodynamic state functions that describe identical chemical events and, ideally, are independent of solution conditions. Further, common metal chromophores used in biological metal sensing studies are proposed as a robust system to determine unknown solution competition.Major conclusionsMetal ions present several complications in ITC experiments. This review presents strategies to avoid these pitfalls and proposes and experimentally validates mathematical approaches to deconvolute complex equilibria that exist in these systems.General significanceThis review discusses the wide range of complications that exists in metal-based ITC experiments. It provides a starting point for scientists new to this field and articulates concerns that will help experienced researchers troubleshoot experiments. This article is part of a Special Issue entitled Microcalorimetry in the BioSciences — Principles and Applications, edited by Fadi Bou-Abdallah.     

Quantitative study of aluminum binding to human serum albumin and transferrin by a chelex competitive binding assay

Binding of aluminum to human serum albumin and transferrin was investigated using a competitive binding assay incorporating a cation exchange resin, chelex. Both albumin and transferrin were found to produce linear Scatchard plots of aluminum binding data over the aluminum and protein concentration ranges found in humans. Binding constants measured for albumin and transferrin were 1.96 and 0.515 microM, respectively.     

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.