Evidence that His349 acts as a pH-inducible switch to accelerate receptor-mediated iron release from the C-lobe of human transferrin

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 (Fe(C)hTF). 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 Fe(C)hTF/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.

Fig. 1

Urea gel analysis of recombinant N-terminal hexa-His-tagged nonglycosylated monoferric human serum transferrin that binds iron only in the C-lobe (Fe_ChTF) and His349 Fe_ChTF mutants. Control and mutant samples were electrophoresed before (iron-containing on the left) and after incubation (on the right) in 100 mM 2-morpholinoethanesulfonic acid (MES) buffer, pH 5.6, containing 300 mM KCl and 4 mM EDTA (apo) for 15 min. a In the absence of the glycosylated, N-terminal hexa-His-tagged soluble recombinant transferrin receptor (residues 121–760) (sTFR) and b in complex with the sTFR. Note that the top band in each lane in b is the sTFR. Because the separation is based on both conformation and charge, the migration patterns of the various mutants differ slightly

Fig. 2

Iron release curves for Fe_ChTF and His349 Fe_ChTF mutants at pH 5.6. Each curve represents an average of three to six experiments. Iron-bound samples (375 nM) in 300 mM KCl were rapidly mixed with 200 mM MES buffer, pH 5.6, containing 300 mM KCl and 8 mM EDTA and excited at 280 nm. The fluorescence emission was monitored by use of a 320-nm cut-on filter. a Black line Fe_ChTF, red line H349A Fe_ChTF, green line H349D Fe_ChTF, pink line H349K Fe_ChTF, blue line H349LFe_ChTF, violet line H349W Fe_ChTF, yellow line H349Y Fe_ChTF. b Black line Fe_ChTF/sTFR, orange line H349A Fe_ChTF/sTFR, green line H349D Fe_ChTF/sTFR, blue line H349L Fe_ChTF/sTFR, violet line H349W Fe_ChTF/sTFR, yellow line H349Y Fe_ChTF/sTFR. Note the difference in x-axis scale

Fig. 3

Kinetic iron release curves and fits for Fe_ChTF and various His349 Fe_ChTF mutants in complex with the sTFR at pH 5.6. a Kinetic curve and fit (red line) of the Fe_ChTF/sTFR complex. b Kinetic curve and fit (red line) of the H349L Fe_ChTF/sTFR complex. c Kinetic curve and fit (red line) of the H349K Fe_ChTF/sTFR complex. d Kinetic curve and fit (red line) of the H349W Fe_ChTF/sTFR complex. Each curve represents an average of three to six runs. Iron-bound samples (375 nM) in 300 mM KCl were rapidly mixed with 200 mM MES buffer, pH 5.6, containing 300 mM KCl and 8 mM EDTA and excited at 280 nm. The fluorescence emission was monitored by use of a 320-nm cut-on filter. The residuals are indicated (green)

Fig. 4

Effect of pH on rate constants from Fe_ChTF/sTFR (a) and H349A Fe_ChTF/sTFR (b) complexes. Rate constants [k₁ for conformational change (black) and k₂ for iron release (white)] as a function of pH are shown for the Fe_ChTF control (a) and the H349A Fe_ChTF mutant (b) in the presence of the sTFR. Except for the pH, the conditions are exactly as indicated in the legend to Fig. 3. Note that the rate constants are plotted on the same scale to provide a direct comparison between the H349A Fe_ChTF/sTFR complex and the Fe_ChTF/sTFR control