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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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TfR Structure(A) Ribbon diagram of TfR homodimer derived from the 3.2 Å structure of TfR (Lawrence et al. 1999). The HFE-binding site (deduced from an analysis using the HFE/TfR co-crystal structure [Bennett et al. 2000]) on the TfR helical domain closest to the viewer is highlighted in cyan.(B) Space-filling representation of one chain from the TfR homodimer, with the HFE structural epitope residues highlighted as in (A). The location of the interdomain cleft is indicated by an orange asterisk.(C–E) Summary of effects of TfR substitutions for binding HFE (C), Fe-Tf (D), and apo-Tf (E). Color-coding of the TfR sidechains designates the effects of the substitutions on binding affinities as indicated.Figures were made with Molscript (Kraulis 1991) or GRASP (Nicholls et al. 1993) and rendered with Raster3D (Merritt and Bacon 1997).
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pbio.0000051-g001: TfR Structure(A) Ribbon diagram of TfR homodimer derived from the 3.2 Å structure of TfR (Lawrence et al. 1999). The HFE-binding site (deduced from an analysis using the HFE/TfR co-crystal structure [Bennett et al. 2000]) on the TfR helical domain closest to the viewer is highlighted in cyan.(B) Space-filling representation of one chain from the TfR homodimer, with the HFE structural epitope residues highlighted as in (A). The location of the interdomain cleft is indicated by an orange asterisk.(C–E) Summary of effects of TfR substitutions for binding HFE (C), Fe-Tf (D), and apo-Tf (E). Color-coding of the TfR sidechains designates the effects of the substitutions on binding affinities as indicated.Figures were made with Molscript (Kraulis 1991) or GRASP (Nicholls et al. 1993) and rendered with Raster3D (Merritt and Bacon 1997).

Mentions: The X-ray crystal structures of the human TfR ectodomain, both alone (Lawrence et al. 1999) and in complex with HFE (Bennett et al. 2000), have been reported. The homodimeric TfR ectodomain contains three domains on each polypeptide chain: a protease-like domain resembling amino- and carboxypeptidases (residues 121–188 and 384–606), an apical domain (residues 189–383), and a helical domain involved in TfR homodimerization (residues 607–760). Intact TfR also includes a glycosylated stalk region (residues 90–120), a transmembrane domain (residues 62–89), and an N-terminal cytoplasmic domain (residues 1–61) that includes a tyrosine-based endosomal sorting sequence (YTRF) (Enns 2002). The structure of a 2:1 HFE/TfR complex (two HFEs bound to a homodimeric TfR) shows that each HFE interacts with helices 1 and 3 of the TfR helical domain (Bennett et al. 2000) (Figure 1A and 1B). The central portion of the interface includes a hydrophobic core consisting of TfR residues Leu619, Val622, and Tyr643 packed against hydrophobic residues from the α1 domain helix of HFE.


Mechanism for multiple ligand recognition by the human transferrin receptor.

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

TfR Structure(A) Ribbon diagram of TfR homodimer derived from the 3.2 Å structure of TfR (Lawrence et al. 1999). The HFE-binding site (deduced from an analysis using the HFE/TfR co-crystal structure [Bennett et al. 2000]) on the TfR helical domain closest to the viewer is highlighted in cyan.(B) Space-filling representation of one chain from the TfR homodimer, with the HFE structural epitope residues highlighted as in (A). The location of the interdomain cleft is indicated by an orange asterisk.(C–E) Summary of effects of TfR substitutions for binding HFE (C), Fe-Tf (D), and apo-Tf (E). Color-coding of the TfR sidechains designates the effects of the substitutions on binding affinities as indicated.Figures were made with Molscript (Kraulis 1991) or GRASP (Nicholls et al. 1993) and rendered with Raster3D (Merritt and Bacon 1997).
© Copyright Policy
Related In: Results  -  Collection

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

pbio.0000051-g001: TfR Structure(A) Ribbon diagram of TfR homodimer derived from the 3.2 Å structure of TfR (Lawrence et al. 1999). The HFE-binding site (deduced from an analysis using the HFE/TfR co-crystal structure [Bennett et al. 2000]) on the TfR helical domain closest to the viewer is highlighted in cyan.(B) Space-filling representation of one chain from the TfR homodimer, with the HFE structural epitope residues highlighted as in (A). The location of the interdomain cleft is indicated by an orange asterisk.(C–E) Summary of effects of TfR substitutions for binding HFE (C), Fe-Tf (D), and apo-Tf (E). Color-coding of the TfR sidechains designates the effects of the substitutions on binding affinities as indicated.Figures were made with Molscript (Kraulis 1991) or GRASP (Nicholls et al. 1993) and rendered with Raster3D (Merritt and Bacon 1997).
Mentions: The X-ray crystal structures of the human TfR ectodomain, both alone (Lawrence et al. 1999) and in complex with HFE (Bennett et al. 2000), have been reported. The homodimeric TfR ectodomain contains three domains on each polypeptide chain: a protease-like domain resembling amino- and carboxypeptidases (residues 121–188 and 384–606), an apical domain (residues 189–383), and a helical domain involved in TfR homodimerization (residues 607–760). Intact TfR also includes a glycosylated stalk region (residues 90–120), a transmembrane domain (residues 62–89), and an N-terminal cytoplasmic domain (residues 1–61) that includes a tyrosine-based endosomal sorting sequence (YTRF) (Enns 2002). The structure of a 2:1 HFE/TfR complex (two HFEs bound to a homodimeric TfR) shows that each HFE interacts with helices 1 and 3 of the TfR helical domain (Bennett et al. 2000) (Figure 1A and 1B). The central portion of the interface includes a hydrophobic core consisting of TfR residues Leu619, Val622, and Tyr643 packed against hydrophobic residues from the α1 domain helix of HFE.

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