The effect of salt concentration on the iron-binding properties of human transferrin.
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ABSTRACT:
SUBMITTER: Williams J
PROVIDER: S-EPMC1163679 | biostudies-other | 1982 Mar
REPOSITORIES: biostudies-other
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Project description: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.
| S-EPMC3267578 | biostudies-literature
Project description:Delivery of iron to cells requires binding of two iron-containing human transferrin (hTF) molecules to the specific homodimeric transferrin receptor (TFR) on the cell surface. Through receptor-mediated endocytosis involving lower pH, salt, and an unidentified chelator, iron is rapidly released from hTF within the endosome. The crystal structure of a monoferric N-lobe hTF/TFR complex (3.22-Å resolution) features two binding motifs in the N lobe and one in the C lobe of hTF. Binding of Fe(N)hTF induces global and site-specific conformational changes within the TFR ectodomain. Specifically, movements at the TFR dimer interface appear to prime the TFR to undergo pH-induced movements that alter the hTF/TFR interaction. Iron release from each lobe then occurs by distinctly different mechanisms: Binding of His349 to the TFR (strengthened by protonation at low pH) controls iron release from the C lobe, whereas displacement of one N-lobe binding motif, in concert with the action of the dilysine trigger, elicits iron release from the N lobe. One binding motif in each lobe remains attached to the same α-helix in the TFR throughout the endocytic cycle. Collectively, the structure elucidates how the TFR accelerates iron release from the C lobe, slows it from the N lobe, and stabilizes binding of apohTF for return to the cell surface. Importantly, this structure provides new targets for mutagenesis studies to further understand and define this system.
| S-EPMC3156180 | biostudies-literature
Project description:The effects of site-directed mutation and salt on the iron(III)-binding site of the recombinant half-molecule of the N-terminal lobe (hTf/2N) of human transferrin was studied by EPR spectroscopy. Changes were observed in the EPR spectra of all variants investigated (D63S, D63C, G65R, K206Q, H207E, H249E, H249Q, K296E and K296Q) compared with that of the wild-type protein. The most pronounced changes in the metal site were caused by replacement of the coordinating residues, Asp-63 and His-249, and the non-coordinating residue Lys-296, which is located in the hinge region of the iron-binding cleft. The EPR spectral changes from replacement of other non-coordinating residues were more subtle, indicating small changes in Fe3+ coordination to the protein. The EPR spectrum of variant G65R suggests that it adopts two distinct conformations in solution, one in which the two domains forming the iron-binding cleft are closed and one in which they are open; in the latter instance Asp-63 is no longer coordinated to the Fe3+. Chloride-binding studies on hTf/2N, K206Q, H207E, K296Q and K296E showed similar binding isotherms, indicating that none of the hinge region residues replaced, i.e. Lys-206, His-207 or Lys-296, are the sites of chloride binding. The results show that the coordination environment of the Fe3+ is sensitive to structural changes from site-directed mutation of both remote and coordinated residues and also to chloride-binding and ionic strength effects.
| S-EPMC1135746 | biostudies-other
Project description:Transferrins belong to an ancient family of extracellular proteins. The best-characterized transferrins are mammalian proteins that function in iron sequestration or iron transport; they accomplish these functions by having a high-affinity iron-binding site in each of their two homologous lobes. Insect hemolymph transferrins (Tsf1s) also function in iron sequestration and transport; however, sequence-based predictions of their iron-binding residues have suggested that most Tsf1s have a single, lower-affinity iron-binding site. To reconcile the apparent contradiction between the known physiological functions and predicted biochemical properties of Tsf1s, we purified and characterized the iron-binding properties of Drosophila melanogaster Tsf1 (DmTsf1), Manduca sexta Tsf1 (MsTsf1), and the amino-lobe of DmTsf1 (DmTsf1N). Using UV-Vis spectroscopy, we found that these proteins bind iron, but they exhibit shifts in their spectra compared to mammalian transferrins. Through equilibrium dialysis experiments, we determined that DmTsf1 and MsTsf1 bind only one ferric ion; their affinity for iron is high (log K' = 18), but less than that of the well-characterized mammalian transferrins (log K' ~ 20); and they release iron under moderately acidic conditions (pH50 = 5.5). Iron release analysis of DmTsf1N suggested that iron binding in the amino-lobe is stabilized by the carboxyl-lobe. These findings will be critical for elucidating the mechanisms of Tsf1 function in iron sequestration and transport in insects.
| S-EPMC7501197 | biostudies-literature
Project description:Recently, we reported a cationic 14 residue peptide LL-14 (LKWLKKLLKWLKKL) with salt-sensitive broad-spectrum antimicrobial potency. However, the mechanism of its salt (NaCl) sensitivity remained unclear. In this study, we have reported computational (∼14.2 μs of MD) and experimental (CD, fluorescence) investigations to examine the salt-sensitivity and the role of peptide secondary structure on LL-14 binding to simple membrane mimetic (SDS, DPC) systems. LL-14 was shown to adopt a random coil (Pc) conformation in water and α-helical conformation (Ph) in the peptide:SDS micelle complex, accompanied by tryptophan burial, using both simulations and experiments. Simulations successfully deconvoluted the LL-14:micelle binding event in terms of secondary structure (random coil Pc versus helix Ph) and gave atomic insight into the initial and final LL-14:SDS complexes. Electrostatics drove the N-terminus (L1 and K2) of LL-14 (Pc or Ph) to bind the SDS micellar surface, initiating complex formation. LL-14 in amphipathic Ph conformation bound faster and buried deeper into the SDS micelle relative to Pc. Increasing NaCl concentration incrementally delayed LL-14:micelle binding by shielding the overall charges of the interacting partners. LL-14 binding to the SDS micelle was significantly faster relative to that of the zwitterionic DPC micelle due to electrostatic reasons. Cationic α-helical amphipathic peptides (with positively charged N-terminus) with low salt-ion concentration seemed to be ideal for faster SDS binding.
| S-EPMC9043568 | biostudies-literature
Project description:Transferrin, the major plasma iron-binding molecule, interacts with cell-surface receptors to deliver iron, modulates hepcidin expression, and regulates erythropoiesis. Transferrin binds and releases iron via either or both of 2 homologous lobes (N and C). To test the hypothesis that the specificity of iron occupancy in the N vs C lobe influences transferrin function, we generated mice with mutations to abrogate iron binding in either lobe (TfN-bl or TfC-bl). Mice homozygous for either mutation had hepatocellular iron loading and decreased liver hepcidin expression (relative to iron concentration), although to different magnitudes. Both mouse models demonstrated some aspects of iron-restricted erythropoiesis, including increased zinc protoporphyrin levels, decreased hemoglobin levels, and microcytosis. Moreover, the TfN-bl/N-bl mice demonstrated the anticipated effect of iron restriction on red cell production (ie, no increase in red blood cell [RBC] count despite elevated erythropoietin levels), along with a poor response to exogenous erythropoietin. In contrast, the TfC-bl/C-bl mice had elevated RBC counts and an exaggerated response to exogenous erythropoietin sufficient to ameliorate the anemia. Observations in heterozygous mice further support a role for relative N vs C lobe iron occupancy in transferrin-mediated regulation of iron homeostasis and erythropoiesis.
| S-EPMC6839954 | biostudies-literature
Project description:AimsTransferrin saturation (TSAT), a marker of iron deficiency, reflects both serum concentrations of iron (SIC) and transferrin (STC). TSAT is susceptible to changes in each of these biomarkers. Little is known about determinants of STC and its influence on TSAT and mortality in patients with heart failure. Accordingly, we studied the relationship of STC to clinical characteristics, to markers of iron deficiency and inflammation and to mortality in chronic heart failure (CHF).Methods and resultsProspective cohort of patients with CHF attending a clinic serving a large local population. A total of 4422 patients were included (median age 75 (68-82) years; 40% women; 32% with left ventricular ejection fraction ≤40%). STC ≤ 2.3 g/L (lowest quartile) was associated with older age, lower SIC and haemoglobin and higher high-sensitivity C-reactive protein, ferritin and N-terminal pro-brain natriuretic peptide compared with those with STC > 2.3 g/L. In the lowest STC quartile, 624 (52%) patients had SIC ≤13 μmol/L, of whom 38% had TSAT ≥20%. For patients in the highest STC quartile, TSAT was <20% when SIC was >13 μmol/L in 185 (17%) patients. STC correlated inversely with ferritin (r = -0.52) and high-sensitivity C-reactive protein (r = -0.17) and directly with albumin (r = 0.29); all P < 0.001. In models adjusted for age, N-terminal pro-brain natriuretic peptide and haemoglobin, both higher SIC (hazard ratio 0.87 [95% CI: 0.81-0.95]) and STC (hazard ratio 0.82 [95% CI: 0.73-0.91]) were associated with lower mortality. SIC was more strongly associated with both anaemia and mortality than either STC or TSAT.ConclusionsMany patients with CHF and a low STC have low SIC even when TSAT is >20% and serum ferritin >100 μg/L; such patients have a high prevalence of anaemia and a poor prognosis and might have iron deficiency but are currently excluded from clinical trials of iron repletion.
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Project description:In order to find out the effect of salt concentration on fermented rape stalks, the physicochemical quality and volatile components was investigated using high performance liquid chromatography (HPLC) and headspace solid phase microextraction gas chromatography-mass spectrometry (HS-SPME-GC-MS). The results showed that there were abundant kinds of free amino acids (FAAs) in all samples, mainly presenting sweet, umami and bitter taste. Through taste activity value (TAV), His, Glu, and Ala contributed significantly to the taste of the sample. 51 volatile components were identified, of which the relative contents of ketones and alcohols were high. By the relative odor activity value (ROAV) analysis, the main components that had a great impact on the flavor were phenylacetaldehyde, β-Ionone, ethyl palmitate and furanone. Adjusting the appropriate salt concentration for fermentation could improve the comprehensive quality of fermented rape stalks and promote the development and utilization of rape products.
| S-EPMC10314211 | biostudies-literature
Project description:The periplasmic iron-binding protein, FbpA (ferric-ion-binding protein A), performs an essential role in iron acquisition from transferrin in Haemophilus influenzae. A series of site-directed mutants in the metal-binding amino acids of FbpA were prepared to determine their relative contribution to iron binding and transport. Structural studies demonstrated that the mutant proteins crystallized in an open conformation with the iron atom associated with the C-terminal domain. The iron-binding properties of the mutant proteins were assessed by several assays, including a novel competitive iron-binding assay. The relative ability of the proteins to compete for iron was pH dependent, with a rank order at pH 6.5 of wild-type, Q58L, H9Q>H9A, E57A>Y195A, Y196A. The genes encoding the mutant FbpA were introduced into H. influenzae and the resulting strains varied in the level of ferric citrate required to support growth on iron-limited medium, suggesting a rank order for metal-binding affinities under physiological conditions comparable with the competitive binding assay at pH 6.5 (wild-type=Q58L>H9Q>H9A, E57A>Y195A, Y196A). Growth dependence on human transferrin was only obtained with cells expressing wild-type, Q58L or H9Q FbpAs, proteins with stability constants derived from the competition assay >2.0x10(18) M(-1). These results suggest that a relatively high affinity of iron binding by FbpA is required for removal of iron from transferrin and its transport across the outer membrane.
| S-EPMC1868806 | biostudies-literature
Project description:Serum transferrin reversibly binds iron in each of two lobes and delivers it to cells by a receptor-mediated, pH-dependent process. The binding and release of iron result in a large conformational change in which two subdomains in each lobe close or open with a rigid twisting motion around a hinge. We report the structure of human serum transferrin (hTF) lacking iron (apo-hTF), which was independently determined by two methods: 1) the crystal structure of recombinant non-glycosylated apo-hTF was solved at 2.7-A resolution using a multiple wavelength anomalous dispersion phasing strategy, by substituting the nine methionines in hTF with selenomethionine and 2) the structure of glycosylated apo-hTF (isolated from serum) was determined to a resolution of 2.7A by molecular replacement using the human apo-N-lobe and the rabbit holo-C1-subdomain as search models. These two crystal structures are essentially identical. They represent the first published model for full-length human transferrin and reveal that, in contrast to family members (human lactoferrin and hen ovotransferrin), both lobes are almost equally open: 59.4 degrees and 49.5 degrees rotations are required to open the N- and C-lobes, respectively (compared with closed pig TF). Availability of this structure is critical to a complete understanding of the metal binding properties of each lobe of hTF; the apo-hTF structure suggests that differences in the hinge regions of the N- and C-lobes may influence the rates of iron binding and release. In addition, we evaluate potential interactions between apo-hTF and the human transferrin receptor.
| S-EPMC1895924 | biostudies-literature