title: Mutational analysis of the transferrin receptor reveals overlapping HFE and transferrin binding sites.
abstract: The transferrin receptor (TfR) binds two proteins critical for iron metabolism: transferrin (Tf) and HFE, the protein mutated in hereditary hemochromatosis. Previous results demonstrated that Tf and HFE compete for binding to TfR, suggesting that Tf and HFE bind to the same or an overlapping site on TfR. TfR is a homodimer that binds one Tf per polypeptide chain (2:2, TfR/Tf stoichiometry), whereas both 2:1 and 2:2 TfR/HFE stoichiometries have been observed. In order to more fully characterize the interaction between HFE and TfR, we determined the binding stoichiometry using equilibrium gel-filtration and analytical ultracentrifugation. Both techniques indicate that a 2:2 TfR/HFE complex can form at submicromolar concentrations in solution, consistent with the hypothesis that HFE competes for Tf binding to TfR by blocking the Tf binding site rather than by exerting an allosteric effect. To determine whether the Tf and HFE binding sites on TfR overlap, residues at the HFE binding site on TfR were identified from the 2.8 A resolution HFE-TfR co-crystal structure, then mutated and tested for their effects on HFE and Tf binding. The binding affinities of soluble TfR mutants for HFE and Tf were determined using a surface plasmon resonance assay. Substitutions of five TfR residues at the HFE binding site (L619A, R629A, Y643A, G647A and F650Q) resulted in significant reductions in Tf binding affinity. The findings that both HFE and Tf form 2:2 complexes with TfR and that mutations at the HFE binding site affect Tf binding support a model in which HFE and Tf compete for overlapping binding sites on TfR.
authors.affiliation: Division of Biology 156-29 , California Institute of Technology, Pasadena, CA 91125, USA.
authors.forename: A P
authors.initials: AP
authors.lastname: West
authors.forename: A M
authors.initials: AM
authors.lastname: Giannetti
authors.forename: A B
authors.initials: AB
authors.lastname: Herr
authors.forename: M J
authors.initials: MJ
authors.lastname: Bennett
authors.forename: J S
authors.initials: JS
authors.lastname: Nangiana
authors.forename: J R
authors.initials: JR
authors.lastname: Pierce
authors.forename: L P
authors.initials: LP
authors.lastname: Weiner
authors.forename: P M
authors.initials: PM
authors.lastname: Snow
authors.forename: P J
authors.initials: PJ
authors.lastname: Bjorkman
mesh_terms: D001667:Binding, Competitive; D002850:Chromatography, Gel; D006680:HLA Antigens / Q000378:metabolism*; D000071020:Hemochromatosis Protein; D015395:Histocompatibility Antigens Class I / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008024:Ligands; D046911:Macromolecular Substances; D008565:Membrane Proteins; D008958:Models, Molecular; D008970:Molecular Weight; D009154:Mutation / Q000235:genetics*; D011487:Protein Conformation; D011990:Receptors, Transferrin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D020349:Surface Plasmon Resonance; D013816:Thermodynamics; D014168:Transferrin / Q000378:metabolism*; D014461:Ultracentrifugation
chemical_list: C103673:HFE protein, human; D006680:HLA Antigens; D000071020:Hemochromatosis Protein; D015395:Histocompatibility Antigens Class I; D008024:Ligands; D046911:Macromolecular Substances; D008565:Membrane Proteins; D011990:Receptors, Transferrin; D014168:Transferrin