Liu Rutao, Guan Jing-Qu, Zak Olga, Aisen Philip, Chance Mark R
Center for Synchrotron Biosciences, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, New York 10461, USA.
Biochemistry. 2003 Nov 4;42(43):12447-54. doi: 10.1021/bi0352973.
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.
人转铁蛋白是一种双叶蛋白，每个叶都有一个单一的铁结合位点，其功能是将铁转运到细胞中。虽然单独的N端叶不能显著结合细胞转铁蛋白受体或作为细胞的铁供体，但C端叶具有这两种功能。我们使用羟基自由基介导的蛋白质足迹法和质谱法来揭示人转铁蛋白C端叶（残基334 - 679）与人类转铁蛋白受体1的胞外域（残基121 - 760）形成复合物时发生的构象变化。通过质谱法测量了C端叶、受体及其复合物中蛋白水解肽的氧化速率；复合物形成后，在对应于残基381 - 401、415 - 433和457 - 470的C端叶序列中，可以看到修饰率显著降低，这表明特定侧链基团受到了保护。C端叶结合后转铁蛋白受体中修饰率降低的肽序列包括残基232 - 240、365 - 371、496 - 508、580和581、614 - 623、634 - 646、647 - 681以及733 - 760。此外，受体中的几个肽表现出修饰率增强，这与复合物形成的变构效应一致。使用串联质谱法，复合物中反应性改变的修饰位点包括C端叶中的Met382、Met389、Trp460、Met464和Phe427以及转铁蛋白受体中的Tyr503、Pro581、Tyr611、Leu619、Met635、Phe650、Trp740、Trp754和Phe760。利用现有的遗传、生化和结构数据，我们证实转铁蛋白受体螺旋结构域中保守的RGD序列（残基646 - 648），包括从Leu619到Phe650的残基，是转铁蛋白C端叶的主要结合位点。
