Structure and membrane binding of alpha-synuclein

在多巴胺能神经元中，a-突触核蛋白（aS）在无序的胞浆状态和脂质结合状态之间分裂。aS与膜磷脂的结合涉及其突触调节的功能作用，但也影响与帕金森病相关的纤维形成。我们在这里描述了一种溶液核磁共振研究，其中将aS添加到模拟突触囊泡的组合物的小单层囊泡中；结果为aS的多种不同磷脂结合模式提供了证据。在核磁共振时间尺度上，自由和脂质结合的aS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的化学计量，并且在低至2:1的化学计量中观察到具有缓慢交换动力学的紧密结合。在所有脂结合状态下，从as的n端开始的残基片段采用a-螺旋构象，而随后的残基保持随机线圈的特征。即使在高脂质浓度下，c端40残基仍保持动态无序，但也可以与脂质结合，其程度似乎取决于该区域顺式X-Pro肽键的比例。虽然脂质结合的aS表现出动力学性质，使其无法通过核磁共振直接观察，但其与核磁共振可见自由形式的交换允许其间接表征。快速的酰胺-酰胺NOE积累指向大的a-螺旋构象，并且Tyr39导致的荧光各向异性的明显增加表明这种暗态的有序环境。随着脂质量的增加，aS的滴定表明，高脂条件下的结合模式在质量上与低脂情况相似。核磁共振数据似乎不符合通常假设的模型，即aS位于膜表面的a-螺旋构象，相反，表明相当大的囊泡重塑是由aS诱导的。

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.