Abstract
His349 in human transferrin (hTF) is a residue critical to transferrin receptor (TFR)-stimulated iron release from the C-lobe. To evaluate the importance of His349 on the TFR interaction, it was replaced by alanine, aspartate, lysine, leucine, tryptophan, and tyrosine in a monoferric C-lobe hTF construct (FeChTF). Using a stopped-flow spectrofluorimeter, we determined rate processes assigned to iron release and conformational events (in the presence and in the absence of the TFR). Significantly, all mutant/TFR complexes feature dampened iron release rates. The critical contribution of His349 is most convincingly revealed by analysis of the kinetics as a function of pH (5.6–6.2). The FeChTF/TFR complex titrates with a pK a of approximately 5.9. By contrast, the H349A mutant/TFR complex releases iron at higher pH with a profile that is almost the inverse of that of the control complex. At the putative endosomal pH of 5.6 (in the presence of salt and chelator), iron is released from the H349W mutant/TFR and H349Y mutant/TFR complexes with a single rate constant similar to the iron release rate constant for the control; this suggests that these substitutions bypass the required pH-induced conformational change allowing the C-lobe to directly interact with the TFR to release iron. The H349K mutant proves that although the positive charge is crucial to complete iron release, the geometry at this position is also critical. The H349D mutant shows that a negative charge precludes complete iron release at pH 5.6 both in the presence and in the absence of the TFR. Thus, histidine uniquely drives the pH-induced conformational change in the C-lobe required for TFR interaction, which in turn promotes iron release.
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Abbreviations
- BHK cells:
-
Baby hamster kidney cells
- Fe2hTF:
-
Diferric human serum transferrin
- FeChTF:
-
Recombinant N-terminal hexa-His-tagged nonglycosylated monoferric human serum transferrin that binds iron only in the C-lobe
- hTF:
-
Human serum transferrin
- MES:
-
2-Morpholinoethanesulfonic acid
- NTA:
-
Nitrilotriacetic acid
- oTF:
-
Ovotransferrin
- sTFR:
-
Glycosylated, N-terminal hexa-His-tagged soluble recombinant transferrin receptor (residues 121–760)
- TBE:
-
Tris(hydroxymethyl)aminomethane–borate–EDTA
- TF:
-
Serum transferrin
- TFR:
-
Transferrin receptor
- Tris:
-
Tris(hydroxymethyl)aminomethane
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Acknowledgments
This work was supported by the United States Public Health Services (R01 DK21739) for A.B.M.and the National Institute of General Medical Sciences (R01 GM20194) for N.D.C. Support for A.N.S. and S.L.B. came from the Hemostasis and Thrombosis Training Grant (5T32HL007594), awarded to K.G. Mann at The University of Vermont by the National Heart, Lung and Blood Institute.
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Steere, A.N., Byrne, S.L., Chasteen, N.D. et al. Evidence that His349 acts as a pH-inducible switch to accelerate receptor-mediated iron release from the C-lobe of human transferrin. J Biol Inorg Chem 15, 1341–1352 (2010). https://doi.org/10.1007/s00775-010-0694-2
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DOI: https://doi.org/10.1007/s00775-010-0694-2