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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 Fe-C-Lobe Binding to Immobilized Wild-Type and Selected Mutant TfR MoleculesPlots of the equilibrium binding response, normalized to the Rmax value (the ligand immobilization value) derived from fitting, versus concentration of injected Fe-C-lobe, are shown for the indicated TfR mutants along with the wild-type TfR control that was present in an adjacent flow cell on the same biosensor chip. Best-fit binding curves derived from a bivalent ligand model are shown as solid lines connecting the datapoints (squares for wild-type TfR and triangles for TfR mutants). The R651A mutant exhibited no binding and was not fit. A summary of derived binding constants is shown in the lower right panel. The KDs for wild-type TfR are averages derived from three independent measurements, and the number after the plus/minus sign represents the standard deviation.
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pbio.0000051-g003: Biosensor Analyses of Fe-C-Lobe Binding to Immobilized Wild-Type and Selected Mutant TfR MoleculesPlots of the equilibrium binding response, normalized to the Rmax value (the ligand immobilization value) derived from fitting, versus concentration of injected Fe-C-lobe, are shown for the indicated TfR mutants along with the wild-type TfR control that was present in an adjacent flow cell on the same biosensor chip. Best-fit binding curves derived from a bivalent ligand model are shown as solid lines connecting the datapoints (squares for wild-type TfR and triangles for TfR mutants). The R651A mutant exhibited no binding and was not fit. A summary of derived binding constants is shown in the lower right panel. The KDs for wild-type TfR are averages derived from three independent measurements, and the number after the plus/minus sign represents the standard deviation.

Mentions: Fe-C-lobe was injected over wild-type TfR and TfR mutants (Y123S, D125K, R651A, F760A) in a biosensor binding assay as described for Fe-Tf above. Binding data were analyzed using an equilibrium-based approach because the rapid kinetics of the Fe-C-lobe interaction with wild-type TfR do not allow accurate derivation of kinetic rate constants (Figure 3). No significant changes in affinity were observed for Fe-C-lobe binding to two mutants in the TfR protease-like domain (Y123S, D125K), but a mutation in the central portion of the Fe-Tf functional epitope on TfR, R651A, eliminated detectable binding (Figure 3). In addition, binding of Fe-C-lobe was not significantly affected by the F760A mutation in the TfR helical domain, which reduces the affinity of apo-Tf, but not Fe-Tf (see Figure 2; Table 1). These results suggest that Tf C-lobe contacts the TfR helical domain, but not the protease-like domain.


Mechanism for multiple ligand recognition by the human transferrin receptor.

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

Biosensor Analyses of Fe-C-Lobe Binding to Immobilized Wild-Type and Selected Mutant TfR MoleculesPlots of the equilibrium binding response, normalized to the Rmax value (the ligand immobilization value) derived from fitting, versus concentration of injected Fe-C-lobe, are shown for the indicated TfR mutants along with the wild-type TfR control that was present in an adjacent flow cell on the same biosensor chip. Best-fit binding curves derived from a bivalent ligand model are shown as solid lines connecting the datapoints (squares for wild-type TfR and triangles for TfR mutants). The R651A mutant exhibited no binding and was not fit. A summary of derived binding constants is shown in the lower right panel. The KDs for wild-type TfR are averages derived from three independent measurements, and the number after the plus/minus sign represents the standard deviation.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC300677&req=5

pbio.0000051-g003: Biosensor Analyses of Fe-C-Lobe Binding to Immobilized Wild-Type and Selected Mutant TfR MoleculesPlots of the equilibrium binding response, normalized to the Rmax value (the ligand immobilization value) derived from fitting, versus concentration of injected Fe-C-lobe, are shown for the indicated TfR mutants along with the wild-type TfR control that was present in an adjacent flow cell on the same biosensor chip. Best-fit binding curves derived from a bivalent ligand model are shown as solid lines connecting the datapoints (squares for wild-type TfR and triangles for TfR mutants). The R651A mutant exhibited no binding and was not fit. A summary of derived binding constants is shown in the lower right panel. The KDs for wild-type TfR are averages derived from three independent measurements, and the number after the plus/minus sign represents the standard deviation.
Mentions: Fe-C-lobe was injected over wild-type TfR and TfR mutants (Y123S, D125K, R651A, F760A) in a biosensor binding assay as described for Fe-Tf above. Binding data were analyzed using an equilibrium-based approach because the rapid kinetics of the Fe-C-lobe interaction with wild-type TfR do not allow accurate derivation of kinetic rate constants (Figure 3). No significant changes in affinity were observed for Fe-C-lobe binding to two mutants in the TfR protease-like domain (Y123S, D125K), but a mutation in the central portion of the Fe-Tf functional epitope on TfR, R651A, eliminated detectable binding (Figure 3). In addition, binding of Fe-C-lobe was not significantly affected by the F760A mutation in the TfR helical domain, which reduces the affinity of apo-Tf, but not Fe-Tf (see Figure 2; Table 1). These results suggest that Tf C-lobe contacts the TfR helical domain, but not the protease-like domain.

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