14 research outputs found
How the binding of human transferrin primes the transferrin receptor potentiating iron release at endosomal pH
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 FeNhTF 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
Inequivalent contribution of the five tryptophan residues in the C-lobe of human serum transferrin to the fluorescence increase when iron is released
Human serum transferrin (hTF), with two Fe(3+) binding lobes transports iron into cells. Diferric hTF preferentially binds to a specific receptor (TFR) on the surface of cells and the complex undergoes clathrin dependent receptor-mediated endocytosis. The clathrin-coated vesicle fuses with an endosome where the pH is lowered, facilitating iron release from hTF. On a biologically relevant timescale (2-3 min), the factors critical to iron release include pH, anions, a chelator and the interaction of hTF with the TFR. Previous work, in which the increase in the intrinsic fluorescence signal was used to monitor iron release from the hTF/TFR complex, established that the TFR significantly enhances the rate of iron release from the C-lobe of hTF. In the current study, the role of the five C-lobe Trp residues in reporting the fluorescence change has been evaluated (± sTFR). Only four of the five recombinant Trp→ Phe mutants produced well. A single slow rate constant for iron release is found for the monoferric C-lobe (Fe(C) hTF) and the four Trp mutants in the Fe(C) hTF background. The three Trp residues equivalent to those in the N-lobe differed from the N-lobe and each other in their contributions to the fluorescent signal. Two rate constants are observed for the Fe(C) hTF control and the four Trp mutants in complex with the TFR: k(obsC1) reports conformational change(s) in the C-lobe initiated by the TFR and k(obsC2) is ascribed to iron release. Excitation at 295 nm (Trp only) and at 280 nm (Trp and Tyr) reveals interesting and significant differences in the rate constants for the complex
Biochemical and structural characterization of recombinant human serum transferrin from rice (Oryza sativa L.)
Structure-Based Mutagenesis Reveals Critical Residues in the Transferrin Receptor Participating in the Mechanism of pH-Induced Release of Iron from Human Serum Transferrin
The recent crystal structure of two monoferric human
serum transferrin (Fe<sub>N</sub>hTF) molecules bound to the soluble
portion of the homodimeric transferrin receptor (sTFR) has provided
new details about this binding interaction that dictates the delivery
of iron to cells. Specifically, substantial rearrangements in the
homodimer interface of the sTFR occur as a result of the binding of
the two Fe<sub>N</sub>hTF molecules. Mutagenesis of selected residues
in the sTFR highlighted in the structure was undertaken to evaluate
the effect on function. Elimination of Ca<sup>2+</sup> binding in
the sTFR by mutating two of four coordinating residues ([E465A,E468A])
results in low production of an unstable and aggregated sTFR. Mutagenesis
of two histidines ([H475A,H684A]) at the dimer interface had little
effect on the kinetics of release of iron at pH 5.6 from either lobe,
reflecting the inaccessibility of this cluster to solvent. Creation
of an H318A sTFR mutant allows assignment of a small pH-dependent
initial decrease in the magnitude of the fluorescence signal to His318.
Removal of the four C-terminal residues of the sTFR, Asp757-Asn758-Glu759-Phe760,
eliminates pH-stimulated release of iron from the C-lobe of the Fe<sub>2</sub>hTF/sTFR Δ757–760 complex. The inability of this
sTFR mutant to bind and stabilize protonated hTF His349 (a pH-inducible
switch) in the C-lobe of hTF accounts for the loss. Collectively,
these studies support a model in which a series of pH-induced events
involving both TFR residue His318 and hTF residue His349 occurs to
promote receptor-stimulated release of iron from the C-lobe of hTF
Structure-Based Mutagenesis Reveals Critical Residues in the Transferrin Receptor Participating in the Mechanism of pH-Induced Release of Iron from Human Serum Transferrin
Structure and dynamics of drug carriers and their interaction with cellular receptors: Focus on serum transferrin
Ionic Residues of Human Serum Transferrin Affect Binding to the Transferrin Receptor and Iron Release
Efficient delivery of iron is critically dependent on
the binding
of diferric human serum transferrin (hTF) to its specific receptor
(TFR) on the surface of actively dividing cells. Internalization of
the complex into an endosome precedes iron removal. The return of
hTF to the blood to continue the iron delivery cycle relies on the
maintenance of the interaction between apohTF and the TFR after exposure
to endosomal pH (≤6.0). Identification of the specific residues
accounting for the pH-sensitive nanomolar affinity with which hTF
binds to TFR throughout the cycle is important to fully understand
the iron delivery process. Alanine substitution of 11 charged hTF
residues identified by available structures and modeling studies allowed
evaluation of the role of each in (1) binding of hTF to the TFR and
(2) TFR-mediated iron release. Six hTF mutants (R50A, R352A, D356A,
E357A, E367A, and K511A) competed poorly with biotinylated diferric
hTF for binding to TFR. In particular, we show that Asp356 in the
C-lobe of hTF is essential to the formation of a stable hTF–TFR
complex: mutation of Asp356 in the monoferric C-lobe hTF background
prevented the formation of the stoichiometric 2:2 (hTF:TFR monomer)
complex. Moreover, mutation of three residues (Asp356, Glu367, and
Lys511), whether in the diferric or monoferric C-lobe hTF, significantly
affected iron release when in complex with the TFR. Thus, mutagenesis
of charged hTF residues has allowed identification of a number of
residues that are critical to formation of and release of iron from
the hTF–TFR complex