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Mutational analysis of the transferrin receptor reveals overlapping HFE and transferrin binding sites

West, Anthony P., Jr. and Giannetti, Anthony M. and Herr, Andrew B. and Bennett, Melanie J. and Nangiana, Jasvinder S. and Pierce, James R. and Weiner, Leslie P. and Snow, Peter M. and Bjorkman, Pamela J. (2001) Mutational analysis of the transferrin receptor reveals overlapping HFE and transferrin binding sites. Journal of Molecular Biology, 313 (2). pp. 385-397. ISSN 0022-2836. https://resolver.caltech.edu/CaltechAUTHORS:20170327-132835015

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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 Å 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.


Item Type:Article
Related URLs:
URLURL TypeDescription
http://dx.doi.org/10.1006/jmbi.2001.5048DOIArticle
http://www.sciencedirect.com/science/article/pii/S002228360195048XPublisherArticle
ORCID:
AuthorORCID
Bjorkman, Pamela J.0000-0002-2277-3990
Additional Information:© 2001 Academic Press. Received 13 July 2001, Revised 28 August 2001, Accepted 28 August 2001, Available online 25 May 2002. We thank D. G. Myszka, José Lebrón, Caroline Enns and W. Lance Martin for helpful discussions, and members of the Bjorkman laboratory for critical reading of the manuscript. A.P.W. was supported by a grant from the Cancer Research Fund of the Damon Runyon-Walter Winchell Foundation Fellowship, DRG-1445. A.M.G. was supported by NRSA predoctoral training grant 5T32-GM07616. A.B.H. was supported by a grant from the Cancer Research Fund of the Damon Runyon-Walter Winchell Foundation Fellowship, DRG-1658. This work was supported by the Howard Hughes Medical Institute and a grant from the Arthritis Foundation (to P.J.B.).
Funders:
Funding AgencyGrant Number
Damon Runyon-Walter Winchell FoundationDRG-1445
NIH Predoctoral Fellowship5T32-GM07616
Damon Runyon-Walter Winchell FoundationDRG-1658
Howard Hughes Medical Institute (HHMI)UNSPECIFIED
Arthritis FoundationUNSPECIFIED
Subject Keywords:HFE; iron overload; surface plasmon resonance; functional epitope; class I MHC homolog
Issue or Number:2
Record Number:CaltechAUTHORS:20170327-132835015
Persistent URL:https://resolver.caltech.edu/CaltechAUTHORS:20170327-132835015
Official Citation:Anthony P. West Jr, Anthony M. Giannetti, Andrew B. Herr, Melanie J. Bennett, Jasvinder S. Nangiana, James R. Pierce, Leslie P. Weiner, Peter M. Snow, Pamela J. Bjorkman, Mutational analysis of the transferrin receptor reveals overlapping HFE and transferrin binding sites1, Journal of Molecular Biology, Volume 313, Issue 2, 19 October 2001, Pages 385-397, ISSN 0022-2836, http://dx.doi.org/10.1006/jmbi.2001.5048. (http://www.sciencedirect.com/science/article/pii/S002228360195048X)
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:75423
Collection:CaltechAUTHORS
Deposited By: Tony Diaz
Deposited On:27 Mar 2017 20:37
Last Modified:03 Oct 2019 16:50

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