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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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Related in: MedlinePlus

Model for the Binding of Fe-Tf and Apo-Tf to TfRThe figures representing each molecule are drawn to scale as an outline around the known structures of TfR (Lawrence et al. 1999), Fe-ovo-Tf (Kurokawa et al. 1995), and apo-ovo-Tf (Kurokawa et al. 1999). Membrane-bound TfR includes a stalk region that places the TfR ectodomain about 30 Å above the cell surface (Fuchs et al. 1998), which would allow the Tf molecule to extend below the plane of the TfR ectodomain. At basic pH, Fe-Tf (orange, with the iron atom positions shown as black dots) and TfR (blue) associate to make a complex containing one TfR homodimer and two Fe-Tf molecules, one bound to each polypeptide chain of the TfR homodimer. Fe-Tf makes energetically favorable contacts at basic pH to residues identified by mutagenesis in the TfR helical domain (red) and the protease-like domain (green). Acidification results in iron release and large conformational changes in the Tf structure as it becomes apo-Tf (gray). Apo-Tf does not make energetically favorable contacts with the protease-like domain, but retains binding to the helical domain-binding site (red) and makes new contacts to the helical domain (yellow), thereby stabilizing the complex. Upon return to basic pH, the apo-Tf molecules dissociate from TfR. This is also illustrated in Video S1.
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pbio.0000051-g004: Model for the Binding of Fe-Tf and Apo-Tf to TfRThe figures representing each molecule are drawn to scale as an outline around the known structures of TfR (Lawrence et al. 1999), Fe-ovo-Tf (Kurokawa et al. 1995), and apo-ovo-Tf (Kurokawa et al. 1999). Membrane-bound TfR includes a stalk region that places the TfR ectodomain about 30 Å above the cell surface (Fuchs et al. 1998), which would allow the Tf molecule to extend below the plane of the TfR ectodomain. At basic pH, Fe-Tf (orange, with the iron atom positions shown as black dots) and TfR (blue) associate to make a complex containing one TfR homodimer and two Fe-Tf molecules, one bound to each polypeptide chain of the TfR homodimer. Fe-Tf makes energetically favorable contacts at basic pH to residues identified by mutagenesis in the TfR helical domain (red) and the protease-like domain (green). Acidification results in iron release and large conformational changes in the Tf structure as it becomes apo-Tf (gray). Apo-Tf does not make energetically favorable contacts with the protease-like domain, but retains binding to the helical domain-binding site (red) and makes new contacts to the helical domain (yellow), thereby stabilizing the complex. Upon return to basic pH, the apo-Tf molecules dissociate from TfR. This is also illustrated in Video S1.

Mentions: Since Tf is a larger molecule than HFE, we reasoned that Tf could also interact with residues not contained in the HFE binding footprint on TfR. We therefore tested substitutions of residues outside of the TfR helical domain for their effects on binding to Tf. To narrow down the search, we chose to substitute solvent-exposed hydrophobic residues, which are often found in protein–protein interfaces (Jones and Thornton 1996; Lo Conte et al. 1999). We also restricted the search to residues within approximately 90 Å (the longest dimension of Fe-Tf) of the Fe-Tf functional-binding epitope for substitution. Using this strategy, we identified a region at the base of the protease-like domain involving residues Tyr123, Trp124, and Asp125, where substitutions showed significant effects on binding to Fe-Tf at pH 7.5, but not to HFE at pH 7.5 or to apo-Tf at pH 6.3 (see Figure 1D and 1E; Table 1). Having defined two predicted Fe-Tf contact areas on TfR that are separated by approximately 33 Å (measured between TfR residues Arg651 and Tyr123) constrains the ways in which Tf can interact with TfR. In particular, computer modeling suggests that a single Tf lobe cannot make productive contacts with both regions of TfR (A. M. Giannetti, unpublished data); thus both lobes of Fe-Tf are likely to be involved in the interface with TfR. Previous studies of the binding of isolated Fe-N- and Fe-C-lobes of Tf suggested that the majority of the binding energy in the Tf/TfR interaction comes from the C-lobe (Zak et al. 1994; Zak and Aisen 2002). It has also been observed that mixing purified N- and C-lobes results in a significant enhancement of TfR binding over that of C-lobe alone (Mason et al. 1997; Zak and Aisen 2002). These observations are consistent with a Tf orientation on TfR in which the C-lobe contacts the Tf functional epitope on the TfR helical domain and the N-lobe contacts the Tyr123 area at the base of the TfR protease-like domain (Figure 4). In this model, allosteric effects need not be invoked to explain the increased affinity of the N-lobe/C-lobe mixture over C-lobe alone (Zak and Aisen 2002). Instead, the observed increase in affinity is predicted to arise from direct contacts between the N-lobe and TfR. To test the predicted orientation of Tf on TfR (Figure 4), we compared the affinities of isolated Fe-C-lobe (Zak and Aisen 2002) to wild-type TfR and to TfR mutants with substitutions in the helical domain (R651A, F760A) and the protease-like domain (Y123S, D125K) (see Figure 3). As predicted, substitutions in the protease-like domain do not affect binding of Fe-C-lobe, whereas a functional epitope substitution (R651A) in the TfR helical domain eliminates detectable binding of Fe-C-lobe to TfR.


Mechanism for multiple ligand recognition by the human transferrin receptor.

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

Model for the Binding of Fe-Tf and Apo-Tf to TfRThe figures representing each molecule are drawn to scale as an outline around the known structures of TfR (Lawrence et al. 1999), Fe-ovo-Tf (Kurokawa et al. 1995), and apo-ovo-Tf (Kurokawa et al. 1999). Membrane-bound TfR includes a stalk region that places the TfR ectodomain about 30 Å above the cell surface (Fuchs et al. 1998), which would allow the Tf molecule to extend below the plane of the TfR ectodomain. At basic pH, Fe-Tf (orange, with the iron atom positions shown as black dots) and TfR (blue) associate to make a complex containing one TfR homodimer and two Fe-Tf molecules, one bound to each polypeptide chain of the TfR homodimer. Fe-Tf makes energetically favorable contacts at basic pH to residues identified by mutagenesis in the TfR helical domain (red) and the protease-like domain (green). Acidification results in iron release and large conformational changes in the Tf structure as it becomes apo-Tf (gray). Apo-Tf does not make energetically favorable contacts with the protease-like domain, but retains binding to the helical domain-binding site (red) and makes new contacts to the helical domain (yellow), thereby stabilizing the complex. Upon return to basic pH, the apo-Tf molecules dissociate from TfR. This is also illustrated in Video S1.
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Related In: Results  -  Collection

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

pbio.0000051-g004: Model for the Binding of Fe-Tf and Apo-Tf to TfRThe figures representing each molecule are drawn to scale as an outline around the known structures of TfR (Lawrence et al. 1999), Fe-ovo-Tf (Kurokawa et al. 1995), and apo-ovo-Tf (Kurokawa et al. 1999). Membrane-bound TfR includes a stalk region that places the TfR ectodomain about 30 Å above the cell surface (Fuchs et al. 1998), which would allow the Tf molecule to extend below the plane of the TfR ectodomain. At basic pH, Fe-Tf (orange, with the iron atom positions shown as black dots) and TfR (blue) associate to make a complex containing one TfR homodimer and two Fe-Tf molecules, one bound to each polypeptide chain of the TfR homodimer. Fe-Tf makes energetically favorable contacts at basic pH to residues identified by mutagenesis in the TfR helical domain (red) and the protease-like domain (green). Acidification results in iron release and large conformational changes in the Tf structure as it becomes apo-Tf (gray). Apo-Tf does not make energetically favorable contacts with the protease-like domain, but retains binding to the helical domain-binding site (red) and makes new contacts to the helical domain (yellow), thereby stabilizing the complex. Upon return to basic pH, the apo-Tf molecules dissociate from TfR. This is also illustrated in Video S1.
Mentions: Since Tf is a larger molecule than HFE, we reasoned that Tf could also interact with residues not contained in the HFE binding footprint on TfR. We therefore tested substitutions of residues outside of the TfR helical domain for their effects on binding to Tf. To narrow down the search, we chose to substitute solvent-exposed hydrophobic residues, which are often found in protein–protein interfaces (Jones and Thornton 1996; Lo Conte et al. 1999). We also restricted the search to residues within approximately 90 Å (the longest dimension of Fe-Tf) of the Fe-Tf functional-binding epitope for substitution. Using this strategy, we identified a region at the base of the protease-like domain involving residues Tyr123, Trp124, and Asp125, where substitutions showed significant effects on binding to Fe-Tf at pH 7.5, but not to HFE at pH 7.5 or to apo-Tf at pH 6.3 (see Figure 1D and 1E; Table 1). Having defined two predicted Fe-Tf contact areas on TfR that are separated by approximately 33 Å (measured between TfR residues Arg651 and Tyr123) constrains the ways in which Tf can interact with TfR. In particular, computer modeling suggests that a single Tf lobe cannot make productive contacts with both regions of TfR (A. M. Giannetti, unpublished data); thus both lobes of Fe-Tf are likely to be involved in the interface with TfR. Previous studies of the binding of isolated Fe-N- and Fe-C-lobes of Tf suggested that the majority of the binding energy in the Tf/TfR interaction comes from the C-lobe (Zak et al. 1994; Zak and Aisen 2002). It has also been observed that mixing purified N- and C-lobes results in a significant enhancement of TfR binding over that of C-lobe alone (Mason et al. 1997; Zak and Aisen 2002). These observations are consistent with a Tf orientation on TfR in which the C-lobe contacts the Tf functional epitope on the TfR helical domain and the N-lobe contacts the Tyr123 area at the base of the TfR protease-like domain (Figure 4). In this model, allosteric effects need not be invoked to explain the increased affinity of the N-lobe/C-lobe mixture over C-lobe alone (Zak and Aisen 2002). Instead, the observed increase in affinity is predicted to arise from direct contacts between the N-lobe and TfR. To test the predicted orientation of Tf on TfR (Figure 4), we compared the affinities of isolated Fe-C-lobe (Zak and Aisen 2002) to wild-type TfR and to TfR mutants with substitutions in the helical domain (R651A, F760A) and the protease-like domain (Y123S, D125K) (see Figure 3). As predicted, substitutions in the protease-like domain do not affect binding of Fe-C-lobe, whereas a functional epitope substitution (R651A) in the TfR helical domain eliminates detectable binding of Fe-C-lobe to TfR.

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