Structure and membrane binding of alpha-synuclein

α-突触核蛋白的结构和膜结合

• 批准号: 7967275
• 负责人: Ad Bax
• 金额: $ 28.11万
• 依托单位: NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7967275
• 关键词: Adopted; Amides; Binding; Biological Process; C-terminal; Characteristics; Data; Disease; Environment; Exhibits; Fluorescence Anisotropy; Kinetics; Lipid Binding; Lipids; Membrane; Modeling; Molecular Conformation; Parkinson Disease; Peptides; Phospholipids; Process; Property; Proteins; Regulation; Role; S-1 Antimetabolite agent; Solutions; Structure; Surface; Synapses; Synaptic Vesicles; Titrations; Vesicle; alpha synuclein; dopaminergic neuron; insight; mimetics; mutant; stoichiometry

In dopaminergic neurons, a-synuclein (aS) partitions between a disordered cytosolic state and a lipid-bound state. Binding of aS to membrane phospholipids is implicated in its functional role of synaptic regulation, but also impacts fibril formation associated with Parkinsons disease. We describe here a solution NMR study in which aS is added to small unilamellar vesicles of a composition mimicking synaptic vesicles; the results provide evidence for multiple distinct phospholipid-binding modes of aS. Exchange between the free and lipid-bound aS state, and between the different bound states, is slow on the NMR timescale, being in the range of 1-10 s-1. Partitioning of the binding modes is dependent on the lipid:aS stoichiometry, and tight binding with slow exchange kinetics is observed at stoichiometries as low as 2:1. In all lipid-bound states, a segment of residues starting at the N-terminus of aSadopts an a-helical conformation while succeeding residues retain characteristics of a random coil. The C-terminal 40 residues remain dynamically disordered, even at high lipid concentration, but can also bind to lipids to an extent that appears to be determined by the fraction of cis X-Pro peptide bonds in this region. While lipid-bound aS exhibits dynamical properties that preclude its direct observation by NMR, its exchange with the NMR-visible free form allows for its indirect characterization. Rapid amide-amide NOE buildup points to a large a-helical conformation, and a distinct increase in fluorescence anisotropy attributed to Tyr39 indicates an ordered environment for this dark state. Titration of aS with increasing amounts of lipids suggests that the binding mode under high lipid conditions remains qualitatively similar to the low-lipid case. The NMR data appear incompatible with the commonly assumed model where aS lies in an a-helical conformation on the membrane surface, and instead suggest that considerable remodeling of the vesicles is induced by aS.

在多巴胺能神经元中，胞质核蛋白（AS）分区之间的胞质状态和脂质结合状态之间的分区。 AS与膜磷脂的结合与其突触调节的功能作用有关，但也影响与帕金森氏病有关的原纤维形成。我们在这里描述了一项溶液NMR研究，其中将其添加到模仿突触囊泡的组成的小囊泡中。结果为AS的多种不同的磷脂结合模式提供了证据。 在NMR时间尺度上，自由和脂质结合的状态和不同结合状态之间的交换在1-10 s-1的范围内速度很慢。 结合模式的划分取决于脂质：作为化学计量学，在较低的2：1处观察到具有缓慢交换动力学的紧密结合。 在所有脂质结合的状态下，从AS AS adpts A a螺旋构象开始的一段残基，而成功的残基保留了随机线圈的特性。 即使在高脂质浓度下，C末端40个残基仍然动态无序，但也可以与脂质结合，这似乎取决于该区域中顺式X-Pro肽键的比例。虽然脂质结合作为表现出的动态特性，这些特性排除了NMR的直接观察，但其与NMR可见的自由形式的交换允许其间接表征。 快速酰胺酰胺NOE的积累表明了大型的A螺旋构象，荧光各向异性归因于Tyr39，这表明该黑暗状态的有序环境。 随着脂质量增加的AS滴定表明，高脂质条件下的结合模式在质量上与低脂情况保持相似。 NMR数据似乎与通常假定的模型不相容，其中如膜表面的A螺旋构象中所致，而是建议对囊泡进行相当大的重塑。