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

Abstract

Delivery of iron to cells requires binding of two iron-containing human transferrin (hTF) molecules to the specific homodimeric transferrin receptor (TFR) on the cell surface. Through receptor-mediated endocytosis involving lower pH, salt, and an unidentified chelator, iron is rapidly released from hTF within the endosome. The crystal structure of a monoferric N-lobe hTF/TFR complex (3.22-Å resolution) features two binding motifs in the N lobe and one in the C lobe of hTF. Binding of Fe(N)hTF induces global and site-specific conformational changes within the TFR ectodomain. Specifically, movements at the TFR dimer interface appear to prime the TFR to undergo pH-induced movements that alter the hTF/TFR interaction. Iron release from each lobe then occurs by distinctly different mechanisms: Binding of His349 to the TFR (strengthened by protonation at low pH) controls iron release from the C lobe, whereas displacement of one N-lobe binding motif, in concert with the action of the dilysine trigger, elicits iron release from the N lobe. One binding motif in each lobe remains attached to the same α-helix in the TFR throughout the endocytic cycle. Collectively, the structure elucidates how the TFR accelerates iron release from the C lobe, slows it from the N lobe, and stabilizes binding of apohTF for return to the cell surface. Importantly, this structure provides new targets for mutagenesis studies to further understand and define this system.

Fig. 1.

Structure of the Fe_NhTF/TFR complex. The biological TFR homodimer (TFR-TFR′, A-A′) with two Fe_NhTF (Fe_NhTF and Fe_NhTF^′, C-C^′) molecules bound is shown oriented with the cell surface at the bottom. The TFR homodimer is colored according to the domains: The apical domain is blue, the protease-like domain is green, and the two monomers of the helical domain are brown and tan. The Ca²⁺ bound within the apical domain of each TFR monomer is shown in yellow. The Fe_NhTF molecules are colored according to subdomain: N1 is gray, N2 is black, and C1 is purple. The bridge between the two lobes is cyan. The Fe³⁺ bound within each N lobe of hTF is shown in red. All figures were prepared using Pymol (43).

Fig. 2.

hTF N-lobe-TFR interaction motifs (also see Table S1). (A) Fe_NhTF residues involved in the N1 interaction motif (gray). Arg50, Tyr68, Tyr71, Ala73, and Asn75 within the N1 subdomain of hTF are close to three residues in the helical domain of the TFR (Gly661, Asn662, and Glu664). Residues Leu72 and Pro74, although involved in the N1 interaction motif, have been omitted for clarity. (B) Fe_NhTF residues involved in the N2 interaction motif (black). The space filling representation of the N2 motif emphasizes that the predominant mode of interaction is van der Waals compared with the H-bonding network for the N1 motif.

Fig. 3.

hTF C1-TFR interaction motif (see Table S1). (A) Residues in the C1 subdomain that contact the TFR are in purple. The space filling representation of the C1 motif emphasizes the predominant van der Waals and packing interactions. The carbonyl oxygen of Gly617 in hTF could hydrogen bond with the ϵ-nitrogen of Arg629 of the TFR (omitted for clarity). Note that critical hTF residue His349 is also not depicted in this representation for enhanced clarity, but is clearly shown in Fig. 4. (B) Comparison of hTF and HFE (22) interactions with TFR αIII-1 and αIII-3 (brown). Secondary structural elements of the C1 subdomain (helix α-1 and strand β-2) that interact with TFR αIII-1 and αIII-3 are shown in purple (as in A). HFE secondary structural elements (helices α-1 and α-2) that interact with TFR αIII-1 and αIII-3 are shown in cyan.

Fig. 4.

Intersection formed between apical domain (blue) and protease-like domain (green) of one TFR monomer (TFR′), the helical domain (brown-tan) of the other TFR monomer (TFR) (Table S2), and the C1 subdomain (purple) of hTF. (A) Our crystal structure of TFR in complex with Fe_NhTF. The maps shown are for the anomalous difference Fourier for the data collected at 0.98 Å contoured at 3 sigma (red) and a simulated annealing composite omit map at 1 sigma (blue). (B) Overlay of A (darker shades) and the cryo-EM complex (1SUV) (24) after least-squares superposition using the TFR molecule (chain A). Secondary structural elements are labeled for clarity. Note that orientation has changed relative to Fig. 1, such that the cell surface is at the top. The Ca²⁺ bound within the apical domain of each TFR monomer is shown in yellow.

Fig. 5.

Plot of the root mean squared deviation calculated using CNS for chain A from the complex compared with a single chain from the receptor alone [red—1CX8 (12)] and when in complex with HFE [black—1DE4 (22)]. (Inset) The table shows the mean rms for both chains of the TFR dimer after superposition of the single chain. P, A, and H refer to the protease-like, apical, and helical domains of the TFR, respectively.