Dollar Ashlyn N, Webb Ian K
Department of Chemistry and Chemical Biology, Indiana University Indianapolis, Indianapolis, Indiana 46202.
Center for Computational Biology and Bioinformatics, Indiana University School of Medicine, Indianapolis, Indiana 46202.
bioRxiv. 2025 Feb 26:2025.02.20.639338. doi: 10.1101/2025.02.20.639338.
Phosphorylation of serine 129 (pS129) in the intrinsically disordered protein alpha synuclein has long been associated with neurodegenerative disease. In the past several years, the functional relevance of pS219 has been uncovered by electrophysiology, immunoprecipitation, and proteomics as intricately connected with neurotransmitter release and synaptic vesicle (SV) cycling. Unexpectedly, binding to SNARE complex proteins VAMP-2 and synapsin only occurs with phosphorylation-competent alpha synuclein. The VAMP-2 binding domain has been shown to be residues 96-110, which does not include the phosphorylated residue, hinting at allosteric regulation of alpha synuclein protein-protein interactions by pS129. Within this study, cross-linking, covalent labeling, and collision induced unfolding of alpha synuclein and pS129 - as well as an additional encountered form in the brain, oxidized-M1, M5, M116, M127 alpha synuclein - are studied utilizing tandem mass spectrometry. Collision induced unfolding of proteins gives a fingerprint of the structures' relative compactness and stabilities of various conformations. Covalent labeling of proteins identifies solvent accessible residues and reveals the hydrophobicity (or hydrophilicity) of their microenvironment, while cross-linking of proteins maps the proximity of residue pairs. The combination of collision induced unfolding, covalent labeling, and cross-linking show unequivocally that phosphorylated-S129 alpha synuclein results in a more stable, more compact form. Our results provide evidence of an extensively folded amphipathic region that interacts strongly with the VAMP-2 binding domain. The phosphorylation-induced folding of the amphipathic region likely tunes other protein-protein interactions and interactions with SVs and membranes.
内在无序蛋白α-突触核蛋白中丝氨酸129（pS129）的磷酸化长期以来一直与神经退行性疾病相关。在过去几年中，pS219的功能相关性已通过电生理学、免疫沉淀和蛋白质组学揭示，其与神经递质释放和突触小泡（SV）循环错综复杂地联系在一起。出乎意料的是，只有具备磷酸化能力的α-突触核蛋白才能与SNARE复合蛋白VAMP-2和突触素结合。VAMP-2结合域已被证明是96 - 110位氨基酸残基，其中不包括磷酸化残基，这暗示了pS129对α-突触核蛋白蛋白质-蛋白质相互作用的变构调节。在本研究中，利用串联质谱研究了α-突触核蛋白和pS129的交联、共价标记以及碰撞诱导展开，以及在大脑中额外发现的一种形式，即氧化型-M1、M5、M116、M127α-突触核蛋白。蛋白质的碰撞诱导展开给出了各种构象结构相对紧凑性和稳定性的指纹图谱。蛋白质的共价标记可识别溶剂可及残基，并揭示其微环境的疏水性（或亲水性），而蛋白质的交联则可绘制残基对的接近程度。碰撞诱导展开、共价标记和交联相结合明确表明，磷酸化的S129α-突触核蛋白形成了一种更稳定、更紧凑的形式。我们的结果提供了一个广泛折叠的两亲区域与VAMP-2结合域强烈相互作用的证据。两亲区域的磷酸化诱导折叠可能会调节其他蛋白质-蛋白质相互作用以及与突触小泡和膜的相互作用。
