Structural reorganization of the transferrin C-lobe and transferrin receptor upon complex formation: the C-lobe binds to the receptor helical domain.
Human transferrin, a bilobal protein, with each lobe bearing a single iron-binding site, functions to transport iron into cells. While the N-terminal lobe alone does not measurably bind cellular transferrin receptors or serve as an iron donor for cells, the C-lobe is capable of both functions. We used hydroxyl radical-mediated protein footprinting and mass spectrometry to reveal the conformational changes that occur upon complex formation for the human transferrin C-lobe (residues 334-679) bound to the ectodomain of human transferrin receptor 1 (residues 121-760). Oxidation rates for proteolytic peptides in the C-lobe, the receptor, and their complex have been measured by mass spectrometry; upon formation of the complex, a dramatic decrease in modification rates, indicating protection of specific side chain groups, can be seen in C-lobe sequences corresponding to residues 381-401, 415-433, and 457-470. Peptide sequences experiencing modification rate decreases in the transferrin receptor upon C-lobe binding include residues 232-240, 365-371, 496-508, 580 and 581, 614-623, 634-646, 647-681, and 733-760. In addition, several peptides in the receptor exhibit enhancements in the rate of modification consistent with allosteric effects of complex formation. Using tandem mass spectrometry, the sites of modification with altered reactivity in the complex include Met382, Met389, Trp460, Met464, and Phe427 in the C-lobe and Tyr503, Pro581, Tyr611, Leu619, Met635, Phe650, Trp740, Trp754, and Phe760 within the transferrin receptor. Using available genetic, biochemical, and structural data, we confirm that the conserved RGD sequence (residues 646-648) in the helical domain of the transferrin receptor, including residues from Leu619 to Phe650, is a primary binding site for the transferrin C-lobe.
Center for Synchrotron Biosciences, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, New York 10461, USA.
Rutao
R
Liu
Jing-Qu
JQ
Guan
Olga
O
Zak
Philip
P
Aisen
Mark R
MR
Chance
D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid; D006801:Humans; D013058:Mass Spectrometry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011485:Protein Binding; D011487:Protein Conformation; D011990:Receptors, Transferrin / Q000737:chemistry* / Q000378:metabolism; D014168:Transferrin / Q000737:chemistry* / Q000378:metabolism
D011990:Receptors, Transferrin; D014168:Transferrin