title: Mechanism for multiple ligand recognition by the human transferrin receptor.
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.
authors.affiliation: Graduate Option in Biochemistry and Molecular Biophysics, California Institute of Technology, Pasadena, California, USA.
authors.forename: Anthony M
authors.initials: AM
authors.lastname: Giannetti
authors.forename: Peter M
authors.initials: PM
authors.lastname: Snow
authors.forename: Olga
authors.initials: O
authors.lastname: Zak
authors.forename: Pamela J
authors.initials: PJ
authors.lastname: Björkman
mesh_terms: D001665:Binding Sites; D000939:Epitopes / Q000737:chemistry; D015723:Gene Library; D006432:Hemochromatosis / Q000378:metabolism; D000071020:Hemochromatosis Protein; D015395:Histocompatibility Antigens Class I / Q000737:chemistry; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007501:Iron / Q000378:metabolism; D008024:Ligands; D008565:Membrane Proteins / Q000737:chemistry; D008956:Models, Chemical; D008969:Molecular Sequence Data; D016296:Mutagenesis; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011990:Receptors, Transferrin / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D012044:Regression Analysis; D020349:Surface Plasmon Resonance; D013816:Thermodynamics; D014168:Transferrin / Q000737:chemistry
chemical_list: D000939:Epitopes; C103673:HFE protein, human; D000071020:Hemochromatosis Protein; D015395:Histocompatibility Antigens Class I; D008024:Ligands; D008565:Membrane Proteins; D011990:Receptors, Transferrin; D014168:Transferrin; D007501:Iron