UVM Theses and Dissertations
Format:
Print
Author:
Steere, Ashley Nicole
Dept./Program:
Biochemistry
Year:
2012
Degree:
PhD
Abstract:
Essential to iron homeostasis is the transport of iron by human serum transferrin (hTF). Each lobe (N-and C-Iobe) of hTF is subdivided into two subdomains (N1 and N2, C1 and C2), which form a deep cleft capable of binding a single ferric iron. Sequestration of Fe³ by hTF serves to prevent its reduction to ferrous iron and potential production of reactive oxygen species via Fenton chemistry. Physiologically, two molecules of ironbearing hTF in the blood (~pH 7.4) bind tightly to the specific transferrin receptor (TFR), a homodimeric transmembrane glycoprotein located on the extracellular surface of dividing cells. At pH 7.4, the TFR preferentially binds diferric hTF (Fe₂hTF) with nanomolar affinity, the two monoferric hTFs (FEnhTF and FechTF) bind with lower affinity (~10 fold), and iron-free (apohTF) does not bind at all. After undergoing clathrindependent endocytosis, the vesicle fuses with an endosome.
Acidification of the endosome initiates the release of Fe³ from hTF in a TFR-mediated process. The subdomains of each lobe undergo large conformational changes in conjunction with iron release to open the cleft: ~60° and ~50° in the N-and C-Iobe, respectively. Significantly, apohTF remains bound to the TFR at acidic pH, in spite of the large conformational changes, implying that the binding parttlers compensate for these structural changes. The apohTF/TFR complex is recycled back to the cell surface, where apohTF is eventually released from the TFR to sequester more iron.
Efficient delivery of iron is critically dependent on the binding of hTF to the TFR. Therefore, identification of the pH-specific contacts accounting for the nanomolar affinity with which hTF binds to the TFR throughout the cycle is important to fully understand the iron delivery process. Recombinant production of both hTF and the soluble portion of the TFR (sTFR) has enabled us to deconvolute this complex system. Use of a stopped-flow spectrofluorimeter has allowed the specific assignment of rate processes to iron release and conformational events from hTF/sTFR complexes. Moreover, a competitive binding assay was utilized to provide a relative binding affinity of hTF mutants in comparison to Fe₂hTF. We have used these methods in an effort to understand the pH-sensitivity of the hTF/TFR interaction and have identified a number of charged hTF residues that are critical to the formation of and iron release from the hTF/TFR complex.
Histidine residues are of particular interest given both the pKa of His residues (~6.0) and the pH range encountered by the hTF/TFR complex during endo-and exocytosis (between pH 7.4-5.6). Analysis of site-directed mutants reveals that protonation of histidines, both in the hTF and the TFR, influence receptor-mediated iron release. Thus, protonation of histidine residues uniquely drives the pH-induced conformational changes required for TFR interaction, which in turn promotes iron release. Collectively, these studies support a model in which pH-induced interrelated events must occur in order to promote receptor-stimulated iron release from hTF.
Acidification of the endosome initiates the release of Fe³ from hTF in a TFR-mediated process. The subdomains of each lobe undergo large conformational changes in conjunction with iron release to open the cleft: ~60° and ~50° in the N-and C-Iobe, respectively. Significantly, apohTF remains bound to the TFR at acidic pH, in spite of the large conformational changes, implying that the binding parttlers compensate for these structural changes. The apohTF/TFR complex is recycled back to the cell surface, where apohTF is eventually released from the TFR to sequester more iron.
Efficient delivery of iron is critically dependent on the binding of hTF to the TFR. Therefore, identification of the pH-specific contacts accounting for the nanomolar affinity with which hTF binds to the TFR throughout the cycle is important to fully understand the iron delivery process. Recombinant production of both hTF and the soluble portion of the TFR (sTFR) has enabled us to deconvolute this complex system. Use of a stopped-flow spectrofluorimeter has allowed the specific assignment of rate processes to iron release and conformational events from hTF/sTFR complexes. Moreover, a competitive binding assay was utilized to provide a relative binding affinity of hTF mutants in comparison to Fe₂hTF. We have used these methods in an effort to understand the pH-sensitivity of the hTF/TFR interaction and have identified a number of charged hTF residues that are critical to the formation of and iron release from the hTF/TFR complex.
Histidine residues are of particular interest given both the pKa of His residues (~6.0) and the pH range encountered by the hTF/TFR complex during endo-and exocytosis (between pH 7.4-5.6). Analysis of site-directed mutants reveals that protonation of histidines, both in the hTF and the TFR, influence receptor-mediated iron release. Thus, protonation of histidine residues uniquely drives the pH-induced conformational changes required for TFR interaction, which in turn promotes iron release. Collectively, these studies support a model in which pH-induced interrelated events must occur in order to promote receptor-stimulated iron release from hTF.