title: Structure-based mutagenesis reveals critical residues in the transferrin receptor participating in the mechanism of pH-induced release of iron from human serum transferrin.
abstract: The recent crystal structure of two monoferric human serum transferrin (Fe(N)hTF) molecules bound to the soluble portion of the homodimeric transferrin receptor (sTFR) has provided new details about this binding interaction that dictates the delivery of iron to cells. Specifically, substantial rearrangements in the homodimer interface of the sTFR occur as a result of the binding of the two Fe(N)hTF molecules. Mutagenesis of selected residues in the sTFR highlighted in the structure was undertaken to evaluate the effect on function. Elimination of Ca(2+) binding in the sTFR by mutating two of four coordinating residues ([E465A,E468A]) results in low production of an unstable and aggregated sTFR. Mutagenesis of two histidines ([H475A,H684A]) at the dimer interface had little effect on the kinetics of release of iron at pH 5.6 from either lobe, reflecting the inaccessibility of this cluster to solvent. Creation of an H318A sTFR mutant allows assignment of a small pH-dependent initial decrease in the magnitude of the fluorescence signal to His318. Removal of the four C-terminal residues of the sTFR, Asp757-Asn758-Glu759-Phe760, eliminates pH-stimulated release of iron from the C-lobe of the Fe(2)hTF/sTFR Δ757-760 complex. The inability of this sTFR mutant to bind and stabilize protonated hTF His349 (a pH-inducible switch) in the C-lobe of hTF accounts for the loss. Collectively, these studies support a model in which a series of pH-induced events involving both TFR residue His318 and hTF residue His349 occurs to promote receptor-stimulated release of iron from the C-lobe of hTF.
authors.affiliation: Department of Biochemistry, University of Vermont, College of Medicine, Burlington, Vermont 05405, United States.
authors.forename: Ashley N
authors.initials: AN
authors.lastname: Steere
authors.forename: N Dennis
authors.initials: ND
authors.lastname: Chasteen
authors.forename: Brendan F
authors.initials: BF
authors.lastname: Miller
authors.forename: Valerie C
authors.initials: VC
authors.lastname: Smith
authors.forename: Ross T A
authors.initials: RT
authors.lastname: MacGillivray
authors.forename: Anne B
authors.initials: AB
authors.lastname: Mason
mesh_terms: D001665:Binding Sites / Q000235:genetics; D002118:Calcium / Q000378:metabolism; D019281:Dimerization; D006801:Humans; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D007501:Iron / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D054730:Protein Interaction Domains and Motifs; D020836:Protein Structure, Quaternary; D011990:Receptors, Transferrin / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014168:Transferrin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D011990:Receptors, Transferrin; D011994:Recombinant Proteins; D014168:Transferrin; D007501:Iron; D002118:Calcium