The chloride effect is related to anion binding in determining the rate of iron release from the human transferrin N-lobe (original) (raw)
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Iron release from recombinant N-lobe and single point Asp63 mutants of human transferrin by EDTA
The Biochemical journal, 1997
Transferrins bind ferric ion and deliver the iron to cells. The mechanism of the iron release has been studied kinetically, in vitro, with the aid of single point mutants in which the iron-binding ligand, Asp63 (aspartic acid-63, D63), has been changed to Ser, Asn, Glu and Ala. Iron release from the unmutated N-lobe of human serum transferrin (hTF/2N) by EDTA is influenced by a variety of factors. The rate-determining conformational-change mechanism may be a major pathway for iron release from hTF/2N's having a 'closed' conformation, which leads to a saturation kinetic mode with respect to ligand concentration. The effect of chloride depends on the protein conformation, showing a negative action in the case of tight binding and a positive action when the protein has an 'open' or 'loose' conformation. The negative effect of chloride could originate from the binding competition between chloride and the chelate to the active site for iron release, and the po...
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 pHsensitive nanomolar affinity with which hTF binds to TFR throughout the cycle is important to fully understand the iron delivery process. Alanine substitution of eleven charged hTF residues identified by available structures and modeling studies allowed evaluation of the role of each in (1) binding of hTF to the TFR and (2) in TFR-mediated iron release. Six hTF mutants (R50A, R352A, D356A, E357A, E367A and K511A) competed poorly with biotinylated diferric hTF for binding to TFR. In particular, we show that Asp356 in the C-lobe of hTF is essential to the formation of a stable hTF/TFR complex: mutation of Asp356 in the monoferric C-lobe hTF background prevented the formation of the stoichiometric 2:2 (hTF:TFR monomer) complex. Moreover, mutation of three residues (Asp356, Glu367 and Lys511), whether in the diferric or monoferric C-lobe hTF, significantly affected iron release when in complex with the TFR. Thus, mutagenesis of charged hTF residues has allowed identification of a number of residues that are critical to formation of and iron release from the hTF/TFR complex.
The Mechanism of Iron Release from the Transferrin-Receptor 1 Adduct
Journal of Molecular Biology, 2006
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%pH%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 Z1.20(G0.05)!10 6 M K1 s K1 and for the C-lobe k 2 Z1.6(G0.1)! 10 3 M K1 s K1. This iron loss is subsequent to a fast proton-driven decarbonation and is followed by two proton gains, (pK 1a)/2Z5.28 per proton for the N-lobe and (pK 2a)/2Z5.10 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 Z1.7(G0.1)!10 K3 s K1. 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 apotransferrinreceptor protein-protein adduct.
Effect of Ligand Structure on the Pathways for Iron Release from Human Serum Transferrin
Inorganic Chemistry, 2005
Rate constants for the removal of iron from N-terminal monoferric transferrin have been measured for a series of phosphate and phosphonocarboxylic acids in pH 7.4 0.1 M hepes buffer at 25°C. The bidentate ligands pyrophosphate and phosphonoacetic acid (PAA) show a combination of saturation and first-order kinetics with respect to the ligand concentration. Similar results are observed following a single substitution at the 2-position of PAA to give 2-benzyl-PAA and phosphonosuccinic acid. In contrast, disubstitution at the 2-position to form 2,2dibenzyl-PAA leads to a marked reduction in iron removal via the first-order pathway. Rate constants were also measured for tripolyphosphate and phosphonodiacetic acid, which are elongated versions of PP i and PAA. In both cases, this elongation completely eliminates the first-order component for iron release while having relatively little impact on the saturation pathway. The sensitivity of the first-order component to the structure of the ligand strongly indicates that this pathway involves the binding of the ligand to a specific site on the protein and cannot be attributed to changes in the overall ionic strength of the solution as the ligand concentration increases. It is proposed that this structural sensitivity reflects steric restrictions on the ability of the incoming ligand to substitute for the synergistic carbonate anion to form a relatively unstable Fe−ligand−Tf ternary intermediate, which then dissociates to FeL and apoTf.
Biochemistry, 2000
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 Å 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.
Anion binding properties of the transferrins. Implications for function
Biochimica et Biophysica Acta (BBA) - General Subjects, 2012
Background: Since the transferrins have been defined by the highly cooperative binding of Fe 3+ and a carbonate anion to form an Fe-CO 3 -Tf ternary complex, the focus has been on synergistic anion binding. However, there are other types of anion binding with both apotransferrin and diferric transferrin that affect metal binding and release. Scope of review: This review covers the binding of anions to the apoprotein, as well as the formation and structure of Fe-anion-transferrin ternary complexes. It also covers interactions between ferric transferrin and non-synergistic anions that appear to be important in vivo. General significance: The interaction of anions with apotransferrin can alter the effective metal binding constants, which can affect the transport of metal ions in serum. These interactions also play a role in iron release under physiological conditions. Major conclusions: Apotransferrin binds a variety of anions with no special selectivity for carbonate. The selectivity for carbonate as a synergistic anion is associated with the iron binding reaction. Conformational changes in the binding of the synergistic carbonate and competition from non-synergistic anions both play a role in intracellular iron release. Anion competition also occurs in serum and reduces the effective metal binding affinity of Tf. Lastly, anions bind to allosteric sites (KISAB sites) on diferric transferrin and alter the rates of iron release. The KISAB sites have not been well-characterized, but kinetic studies on iron release from mutant transferrins indicate that there are likely to be multiple KISAB sites for each lobe of transferrin. This article is part of a Special Issue entitled Transferrins: Molecular mechanisms of iron transport and disorders.
A Kinetically Active Site in the C-Lobe of Human Transferrin †
Biochemistry, 1997
Release of iron from transferrin, the iron-transporting protein of the circulation, is a concerted process involving remote amino acid residues as well as those at the two specific iron-binding sites of the protein. Previous studies of fluoresceinated transferrin have suggested Lys 569 as a kinetically active site in the C-terminal lobe of the protein. We have therefore turned to site-directed mutagenesis to investigate the role of Lys 569 in the release process at pH 5.6, the pH of the endosome where iron is transferred from transferrin to the iron-dependent cell. Mutation of positively charged Lys 569 to an uncharged Gln results in a protein in which release of iron from the mutated lobe to pyrophosphate is slowed by a factor of 15-20 and in which release kinetics switch from a complex saturation-linear to a simple saturation function. Acceleration of release by chloride is also substantially less than in native transferrin. When Lys 569 is replaced by a positively charged Arg, in contrast, observed release rates and chloride dependence are close to those of the native protein. The mechanism of release from the C-lobe site therefore appears to be sensitive to positive charge at position 569. Binding of chloride or other simple anion accelerates and is essential for release from the C-lobe; a muted response of K569Q to chloride concentration suggests that Lys 569 may function as a kinetically active anion-binding residue in the C-lobe. Despite the kinetic effects of the K569 mutation on iron release, rates of iron uptake by K562 cells from the C-lobes of native, K569Q, and K569R proteins are almost identical. In contrast to the C-lobe, iron release from the N-lobe is insensitive to charge at residue 233, the site in that lobe homologous to residue 569, with chloride retarding rather than accelerating release. K233, therefore, is not a kinetically active anion-binding site in the N-lobe. Release mechanisms differ substantially in the two lobes of transferrin despite the identity of ligands and their nearly identical arrangements in the lobes.
FEBS Letters, 2004
2D NMR-pH titrations were used to determine pKa values for four conserved tyrosine residues, Tyr45, Tyr85, Tyr96 and Tyr188 in human transferrin. The low pKa of Tyr188 is due to the fact that the iron-binding ligand interacts with Lys206 in open-form and with Lys296 in the closed-form of the protein. Our current results also confirm the anion binding of sulfate and arsenate to transferrin and further suggest that Tyr188 is the actual binding site for the anions in solution. These data indicate that Tyr188 is a critical residue not only for iron binding but also for chelator binding and iron release in transferrin.
Biochemistry, 2012
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 of this binding interaction which dictates iron delivery to cells. Specifically, substantial rearrangements in the homodimer interface of the sTFR occur as a result of the binding of the two 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 iron release at pH 5.6 from either lobe, reflecting the inaccessibility of this cluster to solvent. Creation of a H318A sTFR mutant allows assignment of a small pH dependent initial decrease in the fluorescent signal to His318. Removal of the four Cterminal residues of the sTFR, Asp757-Asn758-Glu759-Phe760, eliminates pH-stimulated iron release from the C-lobe of the Fe 2 hTF/sTFR Δ757-760 complex. The loss is accounted for by the inability of this sTFR mutant to bind and stabilize protonated hTF His349 (a pH-inducible switch) in the C-lobe of hTF. Collectively, these studies support a model in which a series of pH-induced events involving both TFR residue His318 and hTF residue His349 occurs in order to promote receptor-stimulated iron release from the C-lobe of hTF.