The Synergistic Anion-Binding Sites of Human Transferrin: Chemical and Physiological Effects of Site-Directed Mutagenesis † (original) (raw)
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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.
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.
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.
Journal of Biological Chemistry, 2003
Human serum transferrin (hTF) is a bilobal iron-binding and transport protein that carries iron in the blood stream for delivery to cells by a pH-dependent mechanism. Two iron atoms are held tightly in two deep clefts by coordination to four amino acid residues in each cleft (two tyrosines, a histidine, and an aspartic acid) and two oxygen atoms from the "synergistic" carbonate anion. Other residues in the binding pocket, not directly coordinated to iron, also play critical roles in iron uptake and release through hydrogen bonding to the liganding residues. The original crystal structures of the iron-loaded N-lobe of hTF (pH 5.75 and 6.2) revealed that the synergistic carbonate is stabilized by interaction with Arg-124 and that both the arginine and the carbonate adopt two conformations (MacGillivray, R. . (1998) Biochemistry 37, 7919 -7928). In the present study, we show that the two conformations are also found for a structure at pH 7.7, indicating that this finding was not strictly a function of pH. We also provide structures for two single point mutants (Y45E and L66W) designed to force Arg-124 to adopt each of the previously observed conformations. The structures of each mutant show that this goal was accomplished, and functional studies confirm the hypothesis that access to the synergistic anion dictates the rate of iron release. These studies highlight the importance of the arginine/carbonate movement in the mechanism of iron release in the N-lobe of hTF. Access to the carbonate via a water channel allows entry of protons and anions, enabling the attack on the iron.
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.
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.
The C2 variant of human serum transferrin retains the iron binding properties of the native protein
Biochimica et Biophysica Acta (BBA) - Molecular Basis of Disease, 2005
The tryptic digests of blood samples obtained from transferrin C1 and C2 (TfC1 and TfC2 hereafter) genotypes were analysed by Liquid Chromatography coupled to Electrospray Mass Spectrometry (LC/ESI -MS/MS). The analytical results confirmed the single base change in exon 15 of the Tf gene. The solution behaviour and the iron binding properties of the two Tf variants were studied by UV-visible spectrophotometry and by circular dichroism. It appears that TfC2 globally manifests the same spectral features as the native protein. The local conformation of the two iron binding sites is conserved in the two Tf variants as evidenced by the visible absorption and CD spectra. Also, the iron binding capacities and their pH-dependent profiles are essentially the same. Overall, our investigation points out that the single amino acid substitution in TfC2 (Pro570Ser) does not affect the general conformation of the protein nor the local structure of the iron binding sites. The implications of these results for the etiopathogenesis of Alzheimer's disease are discussed. D
Acta Crystallographica Section D Biological Crystallography, 2007
Iron uptake by humans depends on the ability of the serum protein transferrin (Tf) to bind iron as Fe 3+ with high affinity but reversibly. Iron release into cells occurs through receptormediated endocytosis, aided by the lower endosomal pH of about 5.5. The protonation of a hydrogen-bonded pair of lysines, Lys206 and Lys296, adjacent to the N-lobe iron site of Tf has been proposed to create a repulsive interaction that stimulates domain opening and iron release. The crystal structures of two mutants, K206E (in which Lys206 is mutated to Glu) and K206E/K296E (in which both lysines are mutated to Glu), have been determined. The K206E structure (2.6 Å resolution; R = 0.213, R free = 0.269) shows that a salt bridge is formed between Glu206 and Lys296, thus explaining the drastically slower iron release by this mutant. The K206E/ K296E double-mutant structure (2.8 Å resolution; R = 0.232, R free = 0.259) shows that the Glu296 side chain moves away from Glu206, easing any repulsive interaction and instead interacting with the iron ligand His249. The evident conformational flexibility is consistent with an alternative model for the operation of the dilysine pair in iron release in which it facilitates concerted proton transfer to the tyrosine ligand Tyr188 as one step in the weakening of iron binding.