Partial Metal Ion Saturation of C2 Domains Primes Synaptotagmin 1-Membrane Interactions
Partial Metal Ion Saturation of C2 Domains Primes Synaptotagmin 1-Membrane Interactions
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DOI：
10.1016/j.bpj.2020.01.032
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发表时间：
2020-03-24
影响因子：
3.4
通讯作者：
Igumenova, Tatyana I.
中科院分区：
文献类型：
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作者：
Katti, Sachin;Nyenhuis, Sarah B.;Igumenova, Tatyana I.
Synaptotagmin 1 （Syt1）是一种完整的膜蛋白，其磷脂结合串联C2结构域C2A和C2B作为神经递质释放的Ca2+传感器。我们的目的是了解这些结构域的单个金属离子结合位点在膜结合过程中的作用。我们使用Pb2+， Ca2+的结构和功能替代品，产生具有明确定义的蛋白质-金属离子化学计量学的蛋白质状态。核磁共振实验表明，每个C2结构域结合一个二价金属离子导致环区构象可塑性的丧失，潜在地预先组织它们以进行额外的金属离子和膜结合事件。在C2A中，1位的二价金属离子足以驱动其与含磷脂酰丝氨酸膜的弱结合，而在C2B中，它增强了与信号脂质磷脂酰肌醇-4,5-二磷酸的相互作用。在全长Syt1中，两个Pb2+络合的C2结构域都与含磷脂酰丝氨酸的膜结合。电子顺磁共振实验表明，膜的插入程度与C2金属离子位置的占用有关。总之，我们的研究结果表明，当环内区域的部分金属离子饱和时，Syt1采用动态的部分膜结合状态。这种状态的性质，如构象受限环区和靠近阴离子脂质头基团的C2结构域的定位，“启动”Syt1以协同结合全补体金属离子和更深的膜插入。
Synaptotagmin 1 (Syt1) is an integral membrane protein whose phospholipid-binding tandem C2 domains, C2A and C2B, act as Ca2+ sensors of neurotransmitter release. Our objective was to understand the role of individual metal-ion binding sites of these domains in the membrane association process. We used Pb2+, a structural and functional surrogate of Ca2+, to generate the protein states with well-defined protein-metal ion stoichiometry. NMR experiments revealed that binding of one divalent metal ion per C2 domain results in loss of conformational plasticity of the loop regions, potentially pre-organizing them for additional metal-ion and membrane-binding events. In C2A, a divalent metal ion in site 1 is sufficient to drive its weak association with phosphatidylserine-containing membranes, whereas in C2B, it enhances the interactions with the signaling lipid phosphatidylinositol-4,5-bisphosphate. In full-length Syt1, both Pb2+-complexed C2 domains associate with phosphatidylserine-containing membranes. Electron paramagnetic resonance experiments show that the extent of membrane insertion correlates with the occupancy of the C2 metal ion sites. Together, our results indicate that upon partial metal ion saturation of the intra-loop region, Syt1 adopts a dynamic, partially membrane-bound state. The properties of this state, such as conformationally restricted loop regions and positioning of C2 domains in close proximity to anionic lipid headgroups, "prime'' Syt1 for cooperative binding of a full complement of metal ions and deeper membrane insertion.