Structure and membrane binding of alpha-synuclein

在多巴胺能神经元中，α-突触核蛋白(AS)在无序的胞浆状态和脂质结合状态之间进行划分。AS与膜磷脂的结合不仅涉及其突触调节的功能作用，而且还影响与帕金森病相关的纤维形成。在这里，我们描述了一种溶液核磁共振研究，其中AS被添加到模拟突触小泡的化合物的小单层小泡中；结果为AS的多种不同的磷脂结合模式提供了证据。在核磁共振时间尺度上，游离态和脂结态之间以及不同结合态之间的交换是缓慢的，在1-10 S-1的范围内。结合模式的分配取决于脂质：AS化学计量比，在低至2：1的化学计量比下观察到紧密结合和缓慢的交换动力学。在所有脂质结合状态中，从AS&#61472；N末端开始的一段残基采用a螺旋构象，而随后的残基保持随机卷曲的特征。C-末端40个残基即使在高脂浓度下也保持动态无序，但也可以与脂类结合到似乎由该区域顺式X-Pro多肽键的比例决定的程度。虽然脂质结合的AS表现出动力学性质，排除了它的直接核磁共振观察，但它与核磁共振可见的游离态的交换允许它的间接表征。快速的酰胺-酰胺NOE积聚指向一个大的α-螺旋构象，而由于Tyr39导致的荧光各向异性的显著增加表明这种暗状态的有序环境。随着脂类含量的增加，AS的滴定表明，高脂条件下的结合模式与低脂条件下的结合模式保持定性相似。核磁共振数据似乎与通常假设的模型不相容，即As在膜表面以a-螺旋构象存在，相反，表明As诱导了相当大的囊泡重塑。

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.