Mechanism for Multiple Ligand Recognition by the Human Transferrin Receptor

Transferrin receptor 1 (TfR) plays a critical role in cellular iron import for most higher organisms. Cell surface TfR binds to circulating iron-loaded transferrin (Fe-Tf) and transports it to acidic endosomes, where low pH promotes iron to dissociate from transferrin (Tf) in a TfR-assisted process. The iron-free form of Tf (apo-Tf) remains bound to TfR and is recycled to the cell surface, where the complex dissociates upon exposure to the slightly basic pH of the blood. Fe-Tf competes for binding to TfR with HFE, the protein mutated in the iron-overload disease hereditary hemochromatosis. We used a quantitative surface plasmon resonance assay to determine the binding affinities of an extensive set of site-directed TfR mutants to HFE and Fe-Tf at pH 7.4 and to apo-Tf at pH 6.3. These results confirm the previous finding that Fe-Tf and HFE compete for the receptor by binding to an overlapping site on the TfR helical domain. Spatially distant mutations in the TfR protease-like domain affect binding of Fe-Tf, but not iron-loaded Tf C-lobe, apo-Tf, or HFE, and mutations at the edge of the TfR helical domain affect binding of apo-Tf, but not Fe-Tf or HFE. The binding data presented here reveal the binding footprints on TfR for Fe-Tf and apo-Tf. These data support a model in which the Tf C-lobe contacts the TfR helical domain and the Tf N-lobe contacts the base of the TfR protease-like domain. The differential effects of some TfR mutations on binding to Fe-Tf and apo-Tf suggest differences in the contact points between TfR and the two forms of Tf that could be caused by pH-dependent conformational changes in Tf, TfR, or both. From these data, we propose a structure-based model for the mechanism of TfR-assisted iron release from Fe-Tf

Binding and uptake of H-ferritin are mediated by human transferrin receptor-1

Ferritin is a spherical molecule composed of 24 subunits of two types, ferritin H chain (FHC) and ferritin L chain (FLC). Ferritin stores iron within cells, but it also circulates and binds specifically and saturably to a variety of cell types. For most cell types, this binding can be mediated by ferritin composed only of FHC (HFt) but not by ferritin composed only of FLC (LFt), indicating that binding of ferritin to cells is mediated by FHC but not FLC. By using expression cloning, we identified human transferrin receptor-1 (TfR1) as an important receptor for HFt with little or no binding to LFt. In vitro, HFt can be precipitated by soluble TfR1, showing that this interaction is not dependent on other proteins. Binding of HFt to TfR1 is partially inhibited by diferric transferrin, but it is hindered little, if at all, by HFE. After binding of HFt to TfR1 on the cell surface, HFt enters both endosomes and lysosomes. TfR1 accounts for most, if not all, of the binding of HFt to mitogen-activated T and B cells, circulating reticulocytes, and all cell lines that we have studied. The demonstration that TfR1 can bind HFt as well as Tf raises the possibility that this dual receptor function may coordinate the processing and use of iron by these iron-binding molecules

Model for the Binding of Fe-Tf and Apo-Tf to TfR

<p>The figures representing each molecule are drawn to scale as an outline around the known structures of TfR (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0000051#pbio.0000051-Lawrence1" target="_blank">Lawrence et al. 1999</a>), Fe-ovo-Tf (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0000051#pbio.0000051-Kurokawa1" target="_blank">Kurokawa et al. 1995</a>), and apo-ovo-Tf (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0000051#pbio.0000051-Kurokawa2" target="_blank">Kurokawa et al. 1999</a>). Membrane-bound TfR includes a stalk region that places the TfR ectodomain about 30 Å above the cell surface (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0000051#pbio.0000051-Fuchs1" target="_blank">Fuchs et al. 1998</a>), which would allow the Tf molecule to extend below the plane of the TfR ectodomain. At basic pH, Fe-Tf (orange, with the iron atom positions shown as black dots) and TfR (blue) associate to make a complex containing one TfR homodimer and two Fe-Tf molecules, one bound to each polypeptide chain of the TfR homodimer. Fe-Tf makes energetically favorable contacts at basic pH to residues identified by mutagenesis in the TfR helical domain (red) and the protease-like domain (green). Acidification results in iron release and large conformational changes in the Tf structure as it becomes apo-Tf (gray). Apo-Tf does not make energetically favorable contacts with the protease-like domain, but retains binding to the helical domain-binding site (red) and makes new contacts to the helical domain (yellow), thereby stabilizing the complex. Upon return to basic pH, the apo-Tf molecules dissociate from TfR. This is also illustrated in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0000051#sv001" target="_blank">Video S1</a>.</p

Biosensor Analyses of Fe-C-Lobe Binding to Immobilized Wild-Type and Selected Mutant TfR Molecules

<p>Plots of the equilibrium binding response, normalized to the R_max value (the ligand immobilization value) derived from fitting, versus concentration of injected Fe-C-lobe, are shown for the indicated TfR mutants along with the wild-type TfR control that was present in an adjacent flow cell on the same biosensor chip. Best-fit binding curves derived from a bivalent ligand model are shown as solid lines connecting the datapoints (squares for wild-type TfR and triangles for TfR mutants). The R651A mutant exhibited no binding and was not fit. A summary of derived binding constants is shown in the lower right panel. The K_Ds for wild-type TfR are averages derived from three independent measurements, and the number after the plus/minus sign represents the standard deviation.</p

Biosensor Analyses of Tf Binding to Immobilized Wild-Type and Selected Mutant TfR Molecules

<p>Sensorgrams (black lines) of injected Fe-Tf or apo-Tf binding to wild-type TfR (top left) or the indicated TfR mutants are shown with best-fit binding curves (red lines) derived from a bivalent ligand model (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0000051#s4" target="_blank">Materials and Methods</a>) superimposed. The sensorgrams demonstrate that the binding responses are concentration dependent, and the superimposed binding curves demonstrate the close fit of the binding model to the experimental data. Concentrations of injected proteins for each sensorgram are given below as two numbers: the first is the highest injected concentration (nM), and the second is the dilution factor, either 2-fold (2×) or 3-fold (3×), that relates successive injections. For each TfR sample, there are two sets of numbers, the first being for Fe-Tf and the second for apo-Tf. Wild-type (31, 2×; 200, 2×), Y123S (250, 2×; 330, 3×), W124A (2,000, 3×; 2,000, 2×), D125K (2,000, 3×; 1,000, 2×), W641A (110, 3×; 1,000, 3×), G647A (6,000, 3×; 780, 3×), R651A (5,000, 3×; 1,000, 3×), F760A (110, 3×; 270, 3×).</p

TfR Structure

<div><p>(A) Ribbon diagram of TfR homodimer derived from the 3.2 Å structure of TfR (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0000051#pbio.0000051-Lawrence1" target="_blank">Lawrence et al. 1999</a>). The HFE-binding site (deduced from an analysis using the HFE/TfR co-crystal structure [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0000051#pbio.0000051-Bennett1" target="_blank">Bennett et al. 2000</a>]) on the TfR helical domain closest to the viewer is highlighted in cyan.</p> <p>(B) Space-filling representation of one chain from the TfR homodimer, with the HFE structural epitope residues highlighted as in (A). The location of the interdomain cleft is indicated by an orange asterisk.</p> <p>(C–E) Summary of effects of TfR substitutions for binding HFE (C), Fe-Tf (D), and apo-Tf (E). Color-coding of the TfR sidechains designates the effects of the substitutions on binding affinities as indicated.</p> <p>Figures were made with Molscript (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0000051#pbio.0000051-Kraulis1" target="_blank">Kraulis 1991</a>) or GRASP (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0000051#pbio.0000051-Nicholls1" target="_blank">Nicholls et al. 1993</a>) and rendered with Raster3D (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0000051#pbio.0000051-Merritt1" target="_blank">Merritt and Bacon 1997</a>).</p></div

Immunization for HIV-1 Broadly Neutralizing Antibodies in Human Ig Knockin Mice

A subset of individuals infected with HIV-1 develops broadly neutralizing antibodies (bNAbs) that can prevent infection, but it has not yet been possible to elicit these antibodies by immunization. To systematically explore how immunization might be tailored to produce them, we generated mice expressing the predicted germline or mature heavy chains of a potent bNAb to the CD4 binding site (CD4bs) on the HIV-1 envelope glycoprotein (Env). Immunogens specifically designed to activate B cells bearing germline antibodies are required to initiate immune responses, but they do not elicit bNAbs. In contrast, native-like Env trimers fail to activate B cells expressing germline antibodies but elicit bNAbs by selecting for a restricted group of light chains bearing specific somatic mutations that enhance neutralizing activity. The data suggest that vaccination to elicit anti-HIV-1 antibodies will require immunization with a succession of related immunogen