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Mechanism for multiple ligand recognition by the human transferrin receptor.

Giannetti AM, Snow PM, Zak O, Björkman PJ - PLoS Biol. (2003)

Bottom Line: These results confirm the previous finding that Fe-Tf and HFE compete for the receptor by binding to an overlapping site on the TfR helical domain.The differential effects of some TfR mutations on binding to Fe-Tf and apo-Tf suggest differences in the contact points between TfR and the two forms of Tf that could be caused by pH-dependent conformational changes in Tf, TfR, or both.From these data, we propose a structure-based model for the mechanism of TfR-assisted iron release from Fe-Tf.

View Article: PubMed Central - PubMed

Affiliation: Graduate Option in Biochemistry and Molecular Biophysics, California Institute of Technology, Pasadena, California, USA.

ABSTRACT
Transferrin receptor 1 (TfR) plays a critical role in cellular iron import for most higher organisms. Cell surface TfR binds to circulating iron-loaded transferrin (Fe-Tf) and transports it to acidic endosomes, where low pH promotes iron to dissociate from transferrin (Tf) in a TfR-assisted process. The iron-free form of Tf (apo-Tf) remains bound to TfR and is recycled to the cell surface, where the complex dissociates upon exposure to the slightly basic pH of the blood. Fe-Tf competes for binding to TfR with HFE, the protein mutated in the iron-overload disease hereditary hemochromatosis. We used a quantitative surface plasmon resonance assay to determine the binding affinities of an extensive set of site-directed TfR mutants to HFE and Fe-Tf at pH 7.4 and to apo-Tf at pH 6.3. These results confirm the previous finding that Fe-Tf and HFE compete for the receptor by binding to an overlapping site on the TfR helical domain. Spatially distant mutations in the TfR protease-like domain affect binding of Fe-Tf, but not iron-loaded Tf C-lobe, apo-Tf, or HFE, and mutations at the edge of the TfR helical domain affect binding of apo-Tf, but not Fe-Tf or HFE. The binding data presented here reveal the binding footprints on TfR for Fe-Tf and apo-Tf. These data support a model in which the Tf C-lobe contacts the TfR helical domain and the Tf N-lobe contacts the base of the TfR protease-like domain. The differential effects of some TfR mutations on binding to Fe-Tf and apo-Tf suggest differences in the contact points between TfR and the two forms of Tf that could be caused by pH-dependent conformational changes in Tf, TfR, or both. From these data, we propose a structure-based model for the mechanism of TfR-assisted iron release from Fe-Tf.

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Biosensor Analyses of Tf Binding to Immobilized Wild-Type and Selected Mutant TfR MoleculesSensorgrams (black lines) of injected Fe-Tf or apo-Tf binding to wild-type TfR (top left) or the indicated TfR mutants are shown with best-fit binding curves (red lines) derived from a bivalent ligand model (see Materials and Methods) superimposed. The sensorgrams demonstrate that the binding responses are concentration dependent, and the superimposed binding curves demonstrate the close fit of the binding model to the experimental data. Concentrations of injected proteins for each sensorgram are given below as two numbers: the first is the highest injected concentration (nM), and the second is the dilution factor, either 2-fold (2×) or 3-fold (3×), that relates successive injections. For each TfR sample, there are two sets of numbers, the first being for Fe-Tf and the second for apo-Tf. Wild-type (31, 2×; 200, 2×), Y123S (250, 2×; 330, 3×), W124A (2,000, 3×; 2,000, 2×), D125K (2,000, 3×; 1,000, 2×), W641A (110, 3×; 1,000, 3×), G647A (6,000, 3×; 780, 3×), R651A (5,000, 3×; 1,000, 3×), F760A (110, 3×; 270, 3×).
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pbio.0000051-g002: Biosensor Analyses of Tf Binding to Immobilized Wild-Type and Selected Mutant TfR MoleculesSensorgrams (black lines) of injected Fe-Tf or apo-Tf binding to wild-type TfR (top left) or the indicated TfR mutants are shown with best-fit binding curves (red lines) derived from a bivalent ligand model (see Materials and Methods) superimposed. The sensorgrams demonstrate that the binding responses are concentration dependent, and the superimposed binding curves demonstrate the close fit of the binding model to the experimental data. Concentrations of injected proteins for each sensorgram are given below as two numbers: the first is the highest injected concentration (nM), and the second is the dilution factor, either 2-fold (2×) or 3-fold (3×), that relates successive injections. For each TfR sample, there are two sets of numbers, the first being for Fe-Tf and the second for apo-Tf. Wild-type (31, 2×; 200, 2×), Y123S (250, 2×; 330, 3×), W124A (2,000, 3×; 2,000, 2×), D125K (2,000, 3×; 1,000, 2×), W641A (110, 3×; 1,000, 3×), G647A (6,000, 3×; 780, 3×), R651A (5,000, 3×; 1,000, 3×), F760A (110, 3×; 270, 3×).

Mentions: Each of the TfR mutants designed in the current screen, plus the mutants from the previous study (West et al. 2001), were tested in a surface plasmon resonance-based assay for binding to either a soluble form of HFE at pH 7.5, Fe-Tf at pH 7.5, or apo-Tf at pH 6.3 (Table 1; Figure 2). For these experiments, filtered insect cell supernatants containing secreted recombinant TfR mutants were injected over a biosensor chip to which an anti-pentaHis antibody had been immobilized. The antibody captures TfR by binding to its two 6x-His tags, thereby allowing oriented coupling of the receptors to the biosensor chip. HFE, Fe-Tf, or apo-Tf was then injected over the antibody/TfR-coupled sensor chip, and binding data were fit to a bivalent ligand model in which equilibrium dissociation constants (KD1 and KD2) were derived for binding to the first and the second binding sites on homodimeric TfR (see Table 1) (West et al. 2001).


Mechanism for multiple ligand recognition by the human transferrin receptor.

Giannetti AM, Snow PM, Zak O, Björkman PJ - PLoS Biol. (2003)

Biosensor Analyses of Tf Binding to Immobilized Wild-Type and Selected Mutant TfR MoleculesSensorgrams (black lines) of injected Fe-Tf or apo-Tf binding to wild-type TfR (top left) or the indicated TfR mutants are shown with best-fit binding curves (red lines) derived from a bivalent ligand model (see Materials and Methods) superimposed. The sensorgrams demonstrate that the binding responses are concentration dependent, and the superimposed binding curves demonstrate the close fit of the binding model to the experimental data. Concentrations of injected proteins for each sensorgram are given below as two numbers: the first is the highest injected concentration (nM), and the second is the dilution factor, either 2-fold (2×) or 3-fold (3×), that relates successive injections. For each TfR sample, there are two sets of numbers, the first being for Fe-Tf and the second for apo-Tf. Wild-type (31, 2×; 200, 2×), Y123S (250, 2×; 330, 3×), W124A (2,000, 3×; 2,000, 2×), D125K (2,000, 3×; 1,000, 2×), W641A (110, 3×; 1,000, 3×), G647A (6,000, 3×; 780, 3×), R651A (5,000, 3×; 1,000, 3×), F760A (110, 3×; 270, 3×).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC300677&req=5

pbio.0000051-g002: Biosensor Analyses of Tf Binding to Immobilized Wild-Type and Selected Mutant TfR MoleculesSensorgrams (black lines) of injected Fe-Tf or apo-Tf binding to wild-type TfR (top left) or the indicated TfR mutants are shown with best-fit binding curves (red lines) derived from a bivalent ligand model (see Materials and Methods) superimposed. The sensorgrams demonstrate that the binding responses are concentration dependent, and the superimposed binding curves demonstrate the close fit of the binding model to the experimental data. Concentrations of injected proteins for each sensorgram are given below as two numbers: the first is the highest injected concentration (nM), and the second is the dilution factor, either 2-fold (2×) or 3-fold (3×), that relates successive injections. For each TfR sample, there are two sets of numbers, the first being for Fe-Tf and the second for apo-Tf. Wild-type (31, 2×; 200, 2×), Y123S (250, 2×; 330, 3×), W124A (2,000, 3×; 2,000, 2×), D125K (2,000, 3×; 1,000, 2×), W641A (110, 3×; 1,000, 3×), G647A (6,000, 3×; 780, 3×), R651A (5,000, 3×; 1,000, 3×), F760A (110, 3×; 270, 3×).
Mentions: Each of the TfR mutants designed in the current screen, plus the mutants from the previous study (West et al. 2001), were tested in a surface plasmon resonance-based assay for binding to either a soluble form of HFE at pH 7.5, Fe-Tf at pH 7.5, or apo-Tf at pH 6.3 (Table 1; Figure 2). For these experiments, filtered insect cell supernatants containing secreted recombinant TfR mutants were injected over a biosensor chip to which an anti-pentaHis antibody had been immobilized. The antibody captures TfR by binding to its two 6x-His tags, thereby allowing oriented coupling of the receptors to the biosensor chip. HFE, Fe-Tf, or apo-Tf was then injected over the antibody/TfR-coupled sensor chip, and binding data were fit to a bivalent ligand model in which equilibrium dissociation constants (KD1 and KD2) were derived for binding to the first and the second binding sites on homodimeric TfR (see Table 1) (West et al. 2001).

Bottom Line: These results confirm the previous finding that Fe-Tf and HFE compete for the receptor by binding to an overlapping site on the TfR helical domain.The differential effects of some TfR mutations on binding to Fe-Tf and apo-Tf suggest differences in the contact points between TfR and the two forms of Tf that could be caused by pH-dependent conformational changes in Tf, TfR, or both.From these data, we propose a structure-based model for the mechanism of TfR-assisted iron release from Fe-Tf.

View Article: PubMed Central - PubMed

Affiliation: Graduate Option in Biochemistry and Molecular Biophysics, California Institute of Technology, Pasadena, California, USA.

ABSTRACT
Transferrin receptor 1 (TfR) plays a critical role in cellular iron import for most higher organisms. Cell surface TfR binds to circulating iron-loaded transferrin (Fe-Tf) and transports it to acidic endosomes, where low pH promotes iron to dissociate from transferrin (Tf) in a TfR-assisted process. The iron-free form of Tf (apo-Tf) remains bound to TfR and is recycled to the cell surface, where the complex dissociates upon exposure to the slightly basic pH of the blood. Fe-Tf competes for binding to TfR with HFE, the protein mutated in the iron-overload disease hereditary hemochromatosis. We used a quantitative surface plasmon resonance assay to determine the binding affinities of an extensive set of site-directed TfR mutants to HFE and Fe-Tf at pH 7.4 and to apo-Tf at pH 6.3. These results confirm the previous finding that Fe-Tf and HFE compete for the receptor by binding to an overlapping site on the TfR helical domain. Spatially distant mutations in the TfR protease-like domain affect binding of Fe-Tf, but not iron-loaded Tf C-lobe, apo-Tf, or HFE, and mutations at the edge of the TfR helical domain affect binding of apo-Tf, but not Fe-Tf or HFE. The binding data presented here reveal the binding footprints on TfR for Fe-Tf and apo-Tf. These data support a model in which the Tf C-lobe contacts the TfR helical domain and the Tf N-lobe contacts the base of the TfR protease-like domain. The differential effects of some TfR mutations on binding to Fe-Tf and apo-Tf suggest differences in the contact points between TfR and the two forms of Tf that could be caused by pH-dependent conformational changes in Tf, TfR, or both. From these data, we propose a structure-based model for the mechanism of TfR-assisted iron release from Fe-Tf.

Show MeSH
Related in: MedlinePlus