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与膜磷脂的结合与其突触调节的功能有关，但也影响与帕金森病相关的纤维形成。低浓度的组织学染料刚果红(CR)先前已被证明促进α-突触核蛋白(AS)的聚集，而在较高浓度时，该染料对纤维形成有抑制作用。我们在一系列化学计量比和浓度范围内对pH为6的AS和CR之间的相互作用进行了详细的核磁共振研究，并显示了与先前关于AS与脂泡结合的报道的相似和关键差异。核磁共振弛豫色散测量给出了与CR相互作用的结合分数和时间尺度。当As：Cr摩尔比为1：1时，只有一小部分As(约3%)与CR结合，后者形成较大的(&gt；100 kDa)胶束，但导致As N端60个残基的核磁共振信号强烈衰减。自由态和束缚态之间的快速交换(kex3000 S-1)通过两个过程扩展了As的共振：由自由态和束缚态之间的化学位移差异引起的磁场相关贡献和由As与CR胶束结合的较慢翻滚引起的近场无关的贡献。对与CR相互作用的13Ca二级化学位移的测量表明，如果蛋白质在结合状态下采用纯螺旋几何结构，则相互作用的N-末端残基(1-60)的变化比预期的要小得多，而酰胺-酰胺NOE强度和内部残基与序列HN-Ha NOE的比率表明，在结合状态下，与自由AS相比，伸展结构较少。同时，Ha(I)-HN(I+3)NOE的缺失证实了α-螺旋结构中没有显著的布居。NOE和化学位移数据与异质结合模式兼容，在这种模式下，构象被高度填充。弛豫和化学位移数据表明，在低于约1：1的化学计量比下，AS的NAC区域(包括60-90残基)不参与CR的结合，主要是由于N-末端60个残基的高亲和力竞争CR结合。在As：Cr摩尔比为5：1的情况下，As的NAC区也显示出显著的交换拓宽，分子内顺序和残基内的NO和13Ca化学位移再次表明了一种非均相结合模式，仅显示Ramachandran Phi/psi空间的α区域的布居略有增加。水-LOGSY实验提供了CR和AS之间的分子间NOE接触信息，显示出与N-端30个残基中疏水性较强的子集的接触最强，与40-100个残基的磁化传递较弱，相对均匀的磁化转移到40-100个残基，与C-端35个残基没有相互作用。

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 Parkinson's disease. Low concentrations of the histological dye Congo red (CR) previously have been shown to promote alpha-synuclein (aS) aggregation, whereas at higher levels the dye has an inhibitory effect on fibril formation. We have carried out a detailed NMR study of the interaction between aS and CR at pH 6, over a range of stoichiometries and concentrations, and show similarities as well as key differences relative to prior reports on binding of aS to lipid vesicles. NMR relaxation dispersion measurements yield the bound fraction and time scales for the interaction with CR. At a aS:CR molar ratio of 1:1, only a small fraction of aS (ca 3%) is bound to CR, the latter forming relatively large (> 100 kDa) micelles, but results in strong attenuation of the NMR signals of the N-terminal 60 residues of aS. Rapid exchange (kex 3000 s-1) between the free and the CR-bound state broadens resonances of aS by two processes: a magnetic field dependent contribution, caused by the chemical shift difference between free and bound states, and a nearly field-independent contribution caused by slower tumbling of aS bound to the CR micelle. Measurement of 13Ca secondary chemical shifts upon interaction with CR show changes for the interacting N-terminal residues (1-60) that are much smaller than expected if the protein were to adopt a pure alpha-helical geometry in the bound state, whereas amide-amide NOE intensities and the ratio of intra-residue and sequential HN-Ha NOEs point to less extended structure in the bound state than in free aS. At the same time, the absence of Ha(i)-HN(i+3) NOEs confirms the lack of significant population of alpha-helical structure. NOE and chemical shift data are compatible with a heterogeneous binding mode in which turn conformations are highly populated. Relaxation and chemical shift data indicate that the NAC region of aS (encompassing residues 60-90) is not involved in CR binding at CR:aS stoichiometries lower than ca. 1:1, mostly caused by competition for CR-binding by the higher affinity of the N-terminal 60 residues. At a 5:1 aS:CR molar ratio, the NAC region of aS also shows significant exchange broadening, with intramolecular sequential and intraresidue NOEs and 13Ca chemical shifts indicating again a heterogeneous binding mode that only shows a small increase in population of the alpha region of Ramachandran phi/psi space. A WATER-LOGSY experiment provides intermolecular NOE contact information between CR and aS, showing strongest contacts to a mostly hydrophobic subset of the N-terminal 30 residues and weaker, relatively uniform magnetization transfer to residues 40-100, with no interactions for the C-terminal 35 residues.