The Transferrin Receptor (TfR) - Transferrin (Tf) Complex
)
In
vertebrates, iron is transported in the serum bound to transferrin (Tf),
a glycoprotein of 80 kDa with two homologous lobes (N- and C-lobe). Each
lobe contains two domains (N1, N2 and C1, C2), connected by a flexible
hinge. In the open, iron-free conformation, the two domains are well
separated, while the two domains are closed to coordinate the Fe3+ in
the iron-bearing form. Tf delivers iron to cells using an endocytotic
pathway involving the transferrin receptor (TfR). The structure
of the dimeric TfR ectodomain has been solved by X-ray crystallography,
revealing the butterfly-shaped ectodomain to consist of three domains
(protease-like, apical, and helical domain). At the slightly alkaline
extracellular pH of 7.4, Tf can bind one or two ferric ions, and two
iron-bearing Tf molecules can bind the dimeric TfR; iron-free transferrin
is not recognized by TfR at this pH. The complex is endocytosed, and
the acidic pH of the endosomal lumen induces a conformational change
in Tf that accompanies iron release. The emptied Tf (apoTf) molecules
remain tightly bound to TfR at endosomal pH, and as the complex is returned
to the cell surface the extracellular pH leads to the dissociation of
the apoTf molecules from the receptor.
The Transferrin Receptor - Diferric Transferrin (TfR-dTf) Complex
A model for the TfR-dTf complex was developed
based on the Tf and the TfR ectodomain crystal structures, but
despite significant effort the TfR-dTf complex could not be crystallized.
We have therefore entered a collaboration with Stephen Harrison
(Harvard Medical School) and Philip Aisen (Albert Einstein College
of Medicine) to determine the structure of the human TfR-dTf
complex by cryo-electron microscopy and single particle averaging
techniques. We were able to calculate a density map of the TfR-dTf
complex at sub-nanometer resolution, an unusually high resolution
for single-particle analysis. We were thus able to dock the crystal
structures of the TfR and dTf molecules into our density map
with high accuracy. The resulting model for the complex revealed
an unexpected binding mode for dTf and TfR. The model also shows
a conformational change in Tf induced by association with TfR,
and illustrates the overlap of HFE and Tf binding sites on TfR.
Finally, by replacing the crystal structures of ferric Tf lobes
by those of apoTf lobes, we generated a model for the TfR-apoTf
complex.
) The
density map (gold) obtained by single particle analysis of
vitrified TfR-dTf complex with a nominal resolution of 7.5 Å could
be used to precisely dock the the crystal structures (red)
for the TfR ectodomain and dTf. The missing density for the
apical domain of the TfR ectodomain can be explained by the
flexibility of the linkers that connect this domain to the
remainder of the ectodomain. |
) The
atomic model for the TfR-dTf complex reveals an unexpected
binding interaction between the dTf molecules and the receptor.
Rather than associating with the membrane-distal surfaces
of the receptor, the dTf molecules bind laterally to the
dimeric TfR ectodomain and extend into the gap between the
bulk of the receptor ectodomain and the membrane. The crystal
structures are color-coded; red: TfR protease-like
domain; orange: apical;
yellow: TfR helical domain; light green: Tf N-lobe,
dark green: Tf C-lobe. The TfR stalks (missing in the crystal
structure of the TfR ectodomain) and the membrane surface
are indicated by white lines. |
) Interaction
of Tf C-lobe (left) and Tf N-lobe (right) with the TfR ectodomain
high-lighting the side chains of the residues likely to be
involved in the binding interaction. Only the C1 domain of
the C-lobe interacts with the helical domain of the TfR ectodomain,
whereas both the N1 and the N2 domain of the N-lobe interact
with the receptor. |
) Comparison
of the conformations of receptor-bound (left) and free Tf
(right) reveals a conformational change in the Tf molecule
upon receptor binding. When the orientation of the C-lobe
(dark green) is kept constant, the N-lobe (light green) moves
parallel to the C-lobe by a distance of about 9 Å from
its original position in free Tf (right) to its position
in the complex with the receptor (left). |
) Comparison
of the TfR-dTf complex (top) with the TfR-apoTf complex (bottom),
which was generated by replacing the ferric Tf lobes by apoTf
lobes, shows how the Tf lobes can undergo their opening motion
while remaining bound to the receptor. |
) Modeled
structure of a ternary complex of the TfR dimer (red/orange/yellow)
with one HFE (blue) and one Tf molecule (green) bound. HFE
is the protein mutated in the iron overload disease heredetary
hemochromatosis. The extensive overlap of the Tf and HFE
binding sites on TfR explains how HFE can compete with Tf
for receptor binding. |
 » Cheng et
al. (2004) Cell 116: 565-576. |