Iron release from recombinant N-lobe and single point Asp63 mutants of human transferrin by EDTA (original) (raw)
Related papers
Biochemical Journal, 2000
The major function of human transferrin is to deliver iron from the bloodstream to actively dividing cells. Upon iron release, the protein changes its conformation from ' closed ' to ' open '. Extensive studies in itro indicate that iron release from transferrin is very complex and involves many factors, including pH, the chelator used, an anion effect, temperature, receptor binding and intra-lobe interactions. Our earlier work [He, Biochem. J. 328, 439-445] using the isolated transferrin N-lobe (recombinant N-lobe of human transferrin comprising residues 1-337 ; hTF\2N) has shown that anions and pH modulate iron release from hTF\2N in an interdependent manner : chloride retards iron release at neutral pH, but accelerates the reaction at acidic pH. The present study supports this idea and further details the nature of the dual effect of chloride : the anion effect on iron release is closely related to the strength of anion binding to the apoprotein. The negative effect seems to originate from competition between chloride and the
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.
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.
Structure, 2005
Human transferrin receptor 1 (TfR) binds iron-loaded transferrin (Fe-Tf) and transports it to acidic endosomes where iron is released in a TfR-facilitated process. Consistent with our hypothesis that TfR binding stimulates iron release from Fe-Tf at acidic pH by stabilizing the apo-Tf conformation, a TfR mutant (W641A/ F760A-TfR) that binds Fe-Tf, but not apo-Tf, cannot stimulate iron release from Fe-Tf, and less iron is released from Fe-Tf inside cells expressing W641A/ F760A-TfR than cells expressing wild-type TfR (wtTfR). Electron paramagnetic resonance spectroscopy shows that binding at acidic pH to wtTfR, but not W641A/ F760A-TfR, changes the Tf iron binding site R30 Å from the TfR W641/F760 patch. Mutation of Tf histidine residues predicted to interact with the W641/F760 patch eliminates TfR-dependent acceleration of iron release. Identification of TfR and Tf residues critical for TfR-facilitated iron release, yet distant from a Tf iron binding site, demonstrates that TfR transmits longrange conformational changes and stabilizes the conformation of apo-Tf to accelerate iron release from Fe-Tf.
Proceedings of the National Academy of Sciences, 2003
Human transferrin is a single-chain bilobal protein with each of the two similar but not identical lobes in turn composed of two domains. Each lobe may assume one of two stable structural conformations, open or closed, determined by a rigid rotation of the domains with respect to each other. In solution, the transformation of a lobe between open and closed conformations is associated with the release or binding of an Fe(III) ion. The results of the present study indicate that encapsulation of transferrin within a porous sol-gel matrix allows for a dramatic expansion, to days or weeks, of this interconversion time period, thus providing an opportunity to probe heretofore inaccessible transient intermediates. Sol-gel-encapsulated iron-free transferrin samples are prepared by using two protocols. In the first protocol, the equilibrium form of apotransferrin is encapsulated in the sol-gel matrix, whereas in the second protocol holotransferrin is first encapsulated and then iron is removed from the protein. Results of kinetic and spectroscopic studies allow for distinguishing between two models for iron binding. In the first, iron is assumed to bind to amino acid ligands of one domain, inducing a rigid rotation of the second domain to effect closure of the interdomain cleft. In the second, iron undertakes a conformational search among the thermally accessible states of the lobe, ''choosing'' the state which most nearly approximates the stable closed state when iron is bound. Our experimental results support the second mechanism.
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.
Journal of Molecular Biology, 2010
The 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 of 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 absence of the TFR, unequivocally establishes 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-to 11-fold), and slows the rate of iron release from the N-lobe (6-to 15-fold), making them more equivalent and producing an increase in the net rate of iron removal from Fe 2 hTF. Calculated cooperativity factors, in addition to plots of time dependent species distributions in the absence and 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.
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.
Receptor-modulated iron release from transferrin: differential effects on N- and C-terminal sites
Biochemistry, 1991
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.