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 (FeChTF). Using a stop-ped-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 pKa 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.

How the binding of human transferrin primes the transferrin receptor potentiating iron release at endosomal pH

“…Previous kinetic studies were carried out in a Fe C hTF background (28). Significantly, iron release from this H349A Fe 2 hTF∕TFR complex is preceded by a conformational change with k ¼ 23.7 AE 4.6 min −1 .…”

“…His349 in hTF (Fig. 4), identified as a pH-inducible switch responsible for iron release from the C lobe in the presence of the TFR (27,28), forms the N-terminal cap of hTF helix α-1. At pH 7.5, His349 interacts through both hydrogen bonds and van der Waals interactions with at least two residues in the C-terminal portion of the TFR including Asp757 and Asn758.…”

“…Iron release from the C lobe in the presence of the TFR proceeds by a different mechanism and is 7-to 11-fold faster than in the absence of the TFR (19). Recent studies have demonstrated that iron release from the C lobe is dictated by His349, but only when hTF is bound to the TFR (28). Based on the cryo-EM structure, it was predicted that a hydrophobic patch (TFR residues Trp641 and Phe760) interacts with His349 and stimulates iron release by stabilizing the apohTF/ TFR complex (27).…”

Transferrin Receptors TfR1 and TfR2 Bind Transferrin through Differing Mechanisms

“…There are a number of histidine residues in TfR1 that have been found to be involved in the conformational changes that drive iron release from Tf due to the protonation during endosomal acidification. Further, the Tf residue nearest the junction, His-349, has previously been shown to be essential for release of iron from the C-lobe 36 . His-318 of TfR1 may similarly be required for the transition from a holo-complex to an apo- one, but the stable end states.…”

Transferrin-Mediated Cellular Iron Delivery

“…However, it has been shown that mutation of Lys534 or Arg632 to an alanine severely retards iron release from that lobe, essentially locking iron in the C-lobe (Halbrooks et al, 2003). It has recently become clear that the triad is less relevant to the mechanism of iron release from the C-lobe in the presence of the TFR (Eckenroth, Steere, Chasteen, Everse, & Mason, 2011; Steere, Byrne, et al, 2010) (see discussion of hTF residue His349 below).…”