Myelin protein P2 is a peripheral membrane protein of the fatty acid–binding protein family that functions in the formation and maintenance of the peripheral nerve myelin sheath. Several P2 gene mutations cause human Charcot-Marie-Tooth neuropathy, but the mature myelin sheath assembly mechanism is unclear. Here, cryo-EM of myelin-like proteolipid multilayers revealed an ordered three-dimensional (3D) lattice of P2 molecules between stacked lipid bilayers, visualizing supramolecular assembly at the myelin major dense line. The data disclosed that a single P2 layer is inserted between two bilayers in a tight intermembrane space of ∼3 nm, implying direct interactions between P2 and two membrane surfaces. X-ray diffraction from P2-stacked bicelle multilayers revealed lateral protein organization, and surface mutagenesis of P2 coupled with structure-function experiments revealed a role for both the portal region of P2 and its opposite face in membrane interactions. Atomistic molecular dynamics simulations of P2 on model membrane surfaces suggested that Arg-88 is critical for P2-membrane interactions, in addition to the helical lid domain. Negatively charged lipid headgroups stably anchored P2 on the myelin-like bilayer surface. Membrane binding may be accompanied by opening of the P2 β-barrel structure and ligand exchange with the apposing bilayer. Our results provide an unprecedented view into an ordered, multilayered biomolecular membrane system induced by the presence of a peripheral membrane protein from human myelin. This is an important step toward deciphering the 3D assembly of a mature myelin sheath at the molecular level.

中文翻译：

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低温电磁，X射线衍射和原子模拟揭示了决定因素形成超分子髓鞘样蛋白脂质晶格。

髓磷脂蛋白P2是脂肪酸结合蛋白家族的外周膜蛋白，在外周神经髓鞘的形成和维持中起作用。几种P2基因突变会引起人Charcot-Marie-Tooth神经病，但尚不清楚成熟的髓鞘鞘组装机制。在这里，髓鞘样蛋白脂质多层的冷冻-EM显示堆叠的脂质双层之间的P2分子的有序三维（3D）晶格，可视化髓鞘主要密实线的超分子组装。数据显示，在两个双层之间以〜3 nm的紧密膜间隙插入了一个P2层，这意味着P2和两个膜表面之间存在直接相互作用。从P2堆叠的比塞勒多层膜上进行的X射线衍射揭示了蛋白质的横向组织，P2的表面诱变以及结构功能实验表明，P2的门户区域及其相对面在膜相互作用中均起作用。模型膜表面上P2的原子分子动力学模拟表明，除了螺旋盖结构域外，Arg-88对于P2膜相互作用也至关重要。带负电荷的脂质头基稳定地将P2锚定在髓磷脂样双层表面上。膜结合可能伴随着P2β桶结构的打开和与相对的双层的配体交换。我们的结果为有序的多层生物分子膜系统提供了空前的视野，该系统由人髓磷脂的外周膜蛋白的存在诱导。这是在分子水平上破译成熟髓鞘的3D组装的重要步骤。