Membrane proteins and iron delivery to cells

摘要 我们的长期目标是了解将铁输送到细胞的结构基础。我们尤其 将研究以下三种蛋白质的结构：（/）转铁蛋白（Tf）-转铁蛋白受体（TfR） 复杂.我们已经确定了结构的Tf-TfR复合物使用可溶性结构的TfR 胞外域缺乏柄区。由此产生的结构强烈表明，TfR柄参与 在Tf结合中。我们现在将在茎存在的情况下确定复合体的结构， 功能研究，以阐明TfR茎对铁从受体N-末端叶释放的影响， 结合Tf（//）二价金属离子转运蛋白-1（DMT 1）。从Tf-TfR复合物释放的铁是 通过DMT 1转运穿过内体膜，DMT 1是介导铁吸收的同一种蛋白质， 肠腔通过十二指肠肠上皮细胞的顶面。DMT 1突变导致严重的 低色素性小红细胞贫血和铁超负荷。我们已经表示了DMT 1直系同源物的mg量 来自大肠co//。我们正在使用这种蛋白质来生产适合电子的二维（2D）晶体。 晶体结构测定同时，我们将尝试生长三维（3D）晶体 进行X射线晶体结构测定，并对其他细菌同系物进行结构研究 以及人DMT 1。（Hi）Ferroportin。第二种铁转运蛋白ferroportin将铁出口到整个欧洲。 十二指肠上皮细胞的基底外侧膜与循环的关系。转铁转运蛋白的突变导致IV型 血色素沉着症，也称为膜铁转运蛋白病。我们将表达2D的人膜铁转运蛋白，随后表达2D的人膜铁转运蛋白。 通过电子或X射线晶体学进行3D结晶试验以确定其结构。然后我们将 用肽激素铁调素修饰ferroportin 2D晶体以阐明结合相互作用。 相关性 许多蛋白质依赖于铁作为氧化还原反应或配体配位的辅因子，使铁成为一种蛋白质。 基本元素。亚铁（Fe 2+）和三价铁（Fe 3+）之间的简单转化对铁的形成具有重要意义。 然而，它对活细胞有危险，因为它会导致羟基自由基的形成，这是 对蛋白质、核酸和脂质的氧化损伤。此外，在生理条件下，三价铁 形成高度不溶性的氢氧化物复合物，因此尽管铁含量丰富， 细胞毒性和不溶性迫使高度复杂的机器进化， 储存和分配铁。这些机制的故障会导致缺铁性疾病， 铁超负荷疾病

Abstract Our long-term objective is to understand the structural basis for the delivery of iron to cells. In particular we will study the structure of the following three proteins: (/) The transferrin (Tf)-transferrin receptor (TfR) complex. We have determined the structure of the Tf-TfR complex using a soluble construct of the TfR ectodomain lacking the stalk region. The resulting structure strongly suggests that the TfR stalk is involved in Tf binding. We will now determine the structure of the complex in the presence of the stalk and perform functional studies to elucidate the effect of the TfR stalk on iron release from the N-terminal lobe of receptor- bound Tf. (//) The divalent metal ion transporter-1 (DMT1). Iron released from the Tf-TfR complex is transported across the endosomal membrane by DMT1, the same protein that mediates iron uptake from the intestinal lumen through the apical surface of duodenal enterocytes. Mutations in DMT1 cause severe hypochromic microcytic anemia and iron overload. We have expressed mg amounts of the DMT1 ortholog from E. co//. We are using this protein to produce two-dimensional (2D) crystals suitable for electron crystallographic structure determination. In parallel, we will attempt to grow three-dimensional (3D) crystals for X-ray crystallographic structure determination and perform structural studies on other bacterial homologs as well as human DMT1. (Hi)Ferroportin. A second iron transporter, ferroportin, exports iron across the basolateral membrane of duodenal enterocytes to the circulation. Mutations in ferroportin cause type IV hemochromatosis, also known as ferroportin disease. We will express human ferroportin for 2D and later for 3D crystallization trials to determine its structure either by electron or X-ray crystallography. We will then decorate ferroportin2D crystals with the peptide hormone hepcidin to elucidate the binding interaction. Relevance Many proteins depend on iron as a co-factor for redox reactions or ligand coordination, making iron an essential element. The facile conversion between ferrous (Fe2+) and ferric iron (Fe3+) poses significant dangers to living cells, however, because it can lead to the formation of hydroxyl radicals, a major source for oxidative damage to proteins, nucleic acids and lipids. Moreover, under physiological conditions ferric iron forms a highly insoluble hydroxide complex, so that despite its abundance, iron is not easily accessible to cells. Toxicity and insolubility have forced the evolution of highly sophisticated machineries for acquiring, storing, and distributing iron. Malfunctioning of these machineries lead either to iron deficiency disorders or iron overload diseases.