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Mechanism for Multiple Ligand Recognition by the Human Transferrin Receptor

Giannetti, Anthony M. and Snow, Peter M. and Zak, Olga and Björkman, Pamela J. (2003) Mechanism for Multiple Ligand Recognition by the Human Transferrin Receptor. PLoS Biology, 1 (3). pp. 341-350. ISSN 1544-9173. http://resolver.caltech.edu/CaltechAUTHORS:GIApb03

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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 sitedirected 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.


Item Type:Article
Additional Information:Copyright: © 2003 Giannetti et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Received July 15, 2003; Accepted September 10, 2003; Published December 22, 2003. Supporting Information: Figure S1. [Delta][Delta]G for Mutant TfR Binding to HFE, Fe-Tf, and Apo-Tf Histogram of G values for the change relative to wild-type TfR in TfR mutant affinities for HFE (blue), Fe-Tf (pink), and apo-Tf (gray). G values (the difference in binding energy for a mutant TfR compared to wild-type TfR) were calculated using the KD1 values from Table 1 as G¼RTln(KD1,mut/KD1,wild-type), where R is the gas constant (1.99 3 103 kcal mol1 K1), and T is the temperature in degrees Kelvin (298 K). The dashed green line represents the cutoff for TfR mutants with a greater than or equal to 5-fold affinity reduction in ligand binding, and the dashed red line indicates a greater than or equal to 30-fold affinity reduction. An orange star indicates non-binding mutants and mutants with a greater than 160-fold affinity reduction whose G values exceed the y-axis limit of the histogram (L619A and Y643A, 4 kcal/mol; G647A¼3.2 kcal/mol; and R651A, 4.6 kcal/mol). View online at DOI: 10.1371/journal.pbio.0000051.sg001 (1.86 MB TIFF). Video S1. Model of TfR-Assisted Iron Release from Fe-Tf View online at DOI: 10.1371/journal.pbio.0000051.sv001 (12 MB MOV). Accession Numbers: The SwissProt accessions numbers for the proteins discussed in this paper are b2-microglobulin P01884), Fe-Tf (P02787), HFE (Q30201), TfR canine (Q9GLD3), TfR chicken (Q90997), TfR feline (Q9MYZ3), TfR hamster (Q07891), TfR human (P02786), TfR mouse (Q62351), TfR rat (Q99376), TfR2 human (Q9UP52), and TfR2 mouse (Q62351). Acknowledgments: This work was supported by grants from the National Institutes of Health (1-R01-DK60770 to PJB and DK-15056 to Dr. Philip Aisen) and an National Research Service Award predoctoral training grant (5T32-GM-7616 to AMG). We are grateful to Inderjit Nangiana and Cynthia Jones (Caltech Protein Expression Facility) for assistance in expressing TfR proteins and to the Caltech Protein/Peptide Micro-Analytical Laboratory for protein sequencing. We thank Drs. Anthony West, Andy Herr, Caroline Enns, and Anne B. Mason for helpful discussions and members of the Bjo¨rkman lab for critical reading of the manuscript. We give a special thanks to D. G. Myszka for beta versions of Clamp and Scrubber and discussions of BIACORE experimental details. Conflicts of interest: The authors have declared that no conflicts of interest exist. Author contributions: AMG and PJB conceived and designed the experiments. AMG performed the experiments. AMG and PJB analyzed the data. AMG, PMS, and OZ contributed reagents/materials/analysis tools. AMG and PJB wrote the paper. AMG developed and produced the animated figure.
Subject Keywords:apo-Tf, iron-free transferrin; Fe-C-lobe, iron-loaded transferrin Clobe; Fe-Tf, diferric transferrin; HFE, hereditary hemochromatosis protein; KD, equilibrium dissociation constant; MHC, major histocompatibility complex; PIPES, piperazine-1,4-bis(2-ethanesulphonic) acid; RU, resonance unit; Tf, transferrin; TfR, transferrin receptor
Record Number:CaltechAUTHORS:GIApb03
Persistent URL:http://resolver.caltech.edu/CaltechAUTHORS:GIApb03
Alternative URL:http://dx.doi.org/10.1371/journal.pbio.0000051
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ID Code:282
Collection:CaltechAUTHORS
Deposited By: Archive Administrator
Deposited On:17 May 2005
Last Modified:11 Mar 2014 00:59

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