Abstract：
SLC40A1 is the sole iron export protein reported in mammals. In humans, its dysfunction is responsible for ferroportin disease, an inborn error of iron metabolism transmitted as an autosomal dominant trait and observed in different ethnic groups. As a member of the major facilitator superfamily, SLC40A1 requires a series of conformational changes to enable iron translocation across the plasma membrane. The influence of lipids on protein stability and its conformational changes has been little investigated to date. Here, we combine molecular dynamics simulations of SLC40A1 embedded in membrane bilayers with experimental alanine scanning mutagenesis to analyze the specific role of glycerophospholipids. We identify four basic residues (Lys90, Arg365, Lys366, and Arg371) that are located at the membrane-cytosol interface and consistently interact with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) molecules. These residues surround a network of salt bridges and hydrogens bonds that play a critical role in stabilizing SLC40A1 in its basal outward-facing conformation. More deeply embedded in the plasma membrane, we identify Arg179 as a charged amino acid residue also tightly interacting with lipid polar heads. This results in a local deformation of the lipid bilayer. Interestingly, Arg179 is adjacent to Arg178, which forms a functionally important salt-bridge with Asp473 and is a recurrently associated with ferroportin disease when mutated to glutamine. We demonstrate that the two p.Arg178Gln and p.Arg179Thr missense variants have similar functional behaviors. These observations provide insights into the role of phospholipids in the formation/disruption of the SLC40A1 inner gate, and give a better understanding of the diversity of molecular mechanisms of ferroportin disease.
摘要：
SLC40A1是哺乳动物中报道的唯一的铁输出蛋白。在人类中,它的功能障碍导致了运铁蛋白病，铁代谢的先天性错误以常染色体显性性状传播，并在不同种族中观察到。作为主要主持人超家族的成员，SLC40A1需要一系列构象变化以使铁能够跨质膜易位。迄今为止，很少研究脂质对蛋白质稳定性及其构象变化的影响。这里,我们将嵌入膜双层的SLC40A1的分子动力学模拟与实验丙氨酸扫描诱变相结合，以分析甘油磷脂的具体作用。我们确定了四个碱性残基（Lys90，Arg365，Lys366和Arg371），它们位于膜-细胞质界面，并始终与1-棕榈酰-2-油酰基-sn-甘油-3-磷酸胆碱（POPC）和1-棕榈酰-2-油酰基-sn-甘油-3-磷酸乙醇胺（POPE）分子相互作用。这些残基围绕盐桥和氢键的网络，这些网络在稳定SLC40A1的基础朝外构象中起关键作用。更深地嵌入质膜中，我们确定Arg179为带电荷的氨基酸残基，也与脂质极性头紧密相互作用。这导致脂质双层的局部变形。有趣的是,Arg179与Arg178相邻，后者与Asp473形成功能上重要的盐桥，并且在突变为谷氨酰胺时与铁转运蛋白疾病反复相关。我们证明了两个p.Arg178Gln和p.Arg179Thr错义变体具有相似的功能行为。这些观察结果提供了有关磷脂在SLC40A1内门的形成/破坏中的作用的见解。并更好地了解亚铁转运蛋白病的分子机制的多样性。
