Research Article: Mechanism for Multiple Ligand Recognition by the Human Transferrin Receptor

Date Published: December 22, 2003

Publisher: Public Library of Science

Author(s): Anthony M Giannetti, Peter M Snow, Olga Zak, Pamela J Björkman

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.

Partial Text: Transferrin receptor 1 (TfR) is a homodimeric type II membrane protein that plays a critical role in the primary iron acquisition mechanism for all iron-requiring cell types in vertebrates (Enns 2002). TfR binds the serum iron-carrier protein transferrin (Fe-Tf) and imports it to acidic endosomes, where iron is released and transported to the cytosol. The complex between TfR and iron-free transferrin (apo-Tf) is then recycled to the cell surface where apo-Tf dissociates and returns to circulation (reviewed in Enns et al. 1996). TfR also binds the hereditary hemochromatosis protein HFE (Parkkila et al. 1997; Feder et al. 1998). HFE is a class I major histocompatibility complex (MHC)-related protein that is mutated in patients with hereditary hemochromatosis (Feder et al. 1996), an iron-storage disease characterized by excessive iron absorption leading to an accumulation of iron principally in the liver, heart, pancreas, parathyroid, and pituitary gland, leading to tissue damage (Cullen et al. 1999).

Despite many years of investigation of the Tf/TfR pathway for iron uptake, molecular details about the interaction between TfR and Tf have been limited largely due to a lack of structural information for a Tf/TfR complex. In the absence of a three-dimensional structure, site-directed mutagenesis can be used to map out a protein–protein interaction. To narrow down a subset of residues for mutageneis from the 639 residues in a soluble TfR monomer, we used the crystal structures of TfR alone (Lawrence et al. 1999) and TfR bound to HFE (Bennett et al. 2000) to locate solvent-exposed residues in the vicinity of the HFE-binding site, which was suggested from competition studies to overlap with the Tf-binding site on TfR (Lebrón et al. 1999). We identified residues within the TfR helical domain whose substitution affected binding of both HFE and Fe-Tf at pH 7.5 in a previous mutagenesis study involving ten human TfR mutants (West et al. 2001). These results established that HFE and Fe-Tf bind to the same or an overlapping site on TfR. In the present study, we have expanded the library of TfR mutants to more precisely map the Tf-binding site on TfR and compared binding of Fe-Tf and apo-Tf to TfR. From a survey of 29 point mutants of human TfR, we identified 11 residues, which, when substituted, reduce the affinity of TfR for either human Fe-Tf, apo-Tf, or both (see Table 1). Six of the 11 residues are completely conserved in different species of TfR and in a more recently identified Tf-binding receptor, TfR2, which shares 45% sequence identity with TfR (Kawabata et al. 1999). Most notably, four of the residues exerting the largest effects on Fe-Tf binding, apo-Tf binding, or both (Leu619, Trp641, Gly647, and Arg651) are completely conserved across all currently known TfR and TfR2 sequences (see Table 1). Others, such as the tyrosines at positions 123 and 643, are either conserved or conservatively substituted for phenylalanine in some TfR species. By contrast, of the 18 positions at which substitutions did not significantly affect Tf binding, 16 are not conserved, and two (Phe187 and Glu664) are conservatively substituted (see Table 1). These results suggest that our conclusions about the mode of binding between human Tf and human TfR can be generalized to include Tf/TfR complexes from other species and the interaction between TfR2 and Tf.



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