Abstract
The structural allostery and binding interface for the human serum transferrin (Tf)*transferrin receptor (TfR) complex were identified using radiolytic footprinting and mass spectrometry. We have determined previously that the transferrin C-lobe binds to the receptor helical domain. In this study we examined the binding interactions of full-length transferrin with receptor and compared these data with a model of the complex derived from cryoelectron microscopy (cryo-EM) reconstructions (Cheng, Y., Zak, O., Aisen, P., Harrison, S. C. & Walz, T. (2004) Structure of the human transferrin receptor.transferrin complex. Cell 116, 565-576). The footprinting results provide the following novel conclusions. First, we report characteristic oxidations of acidic residues in the C-lobe of native Tf and basic residues in the helical domain of TfR that were suppressed as a function of complex formation; this confirms ionic interactions between these protein segments as predicted by cryo-EM data and demonstrates a novel method for detecting ion pair interactions in the formation of macromolecular complexes. Second, the specific side-chain interactions between the C-lobe and N-lobe of transferrin and the corresponding interactions sites on the transferrin receptor predicted from cryo-EM were confirmed in solution. Last, the footprinting data revealed allosteric movements of the iron binding C- and N-lobes of Tf that sequester iron as a function of complex formation; these structural changes promote tighter binding of the metal ion and facilitate efficient ion transport during endocytosis.
Highlights
The structural allostery and binding interface for the human serum transferrin (Tf)1⁄7transferrin receptor (TfR) complex were identified using radiolytic footprinting and mass spectrometry
Potential ion pair interactions between acidic residues in the Tf-C lobe and basic residues in the TfR helical domain that might mediate both specific as well as high affinity C-lobe binding to the helical domain were suggested (3, 9 –11)
We compare these model predictions with the footprinting data shown in Fig. 2, A and B, to evaluate the contacts predicted by the model and to identify regions of potential allosteric change as a function of complex formation
Summary
The structural allostery and binding interface for the human serum transferrin (Tf)1⁄7transferrin receptor (TfR) complex were identified using radiolytic footprinting and mass spectrometry. We report characteristic oxidations of acidic residues in the C-lobe of native Tf and basic residues in the helical domain of TfR that were suppressed as a function of complex formation; this confirms ionic interactions between these protein segments as predicted by cryo-EM data and demonstrates a novel method for detecting ion pair interactions in the formation of macromolecular complexes. Cheng et al [3] obtained an atomic model of the human Tf1⁄7TfR complex by docking the crystal structures of human TfR ectodomain and iron-loaded N-terminal and C-terminal lobes of Tf into a 7.5-Å resolution density map generated by cryoelectron microscopy (cryo-EM) image reconstruction and single particle averaging techniques. The cryo-EM data are just suggestive with respect to the specific side chains involved, and methods to confirm protein/ligand interfaces and their charge-charge interactions are essential for understanding molecular recognition involving the specific and dynamic interactions of biological assemblies
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
