Crystal structure of the RIM2 C2A-domain at 1.4 Å resolution
Crystal structure of the RIM2 C2A-domain at 1.4 Å resolution
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DOI：
10.1021/bi0513608
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发表时间：
2005-10-18
期刊：
影响因子：
2.9
通讯作者：
Rizo, J
中科院分区：
文献类型：
--
作者：
Dai, H;Tomchick, DR;Rizo, J
rim是含有两个c -2结构域的大蛋白质，位于突触前活跃区，神经递质在此释放。环在突触囊泡启动和突触前可塑性调节中起关键作用。RIM1 C(2)A结构域的突变与常染色体显性锥杆营养不良（CORD7）有关。RIM c2a结构域不包含通常介导Ca2+在c2结构域顶部环结合的天冬氨酸残基的全部补体，并且已报道与SNAP-25和synaptotagmin 1相互作用，这两种蛋白来自Ca2+依赖的膜融合机制。本文利用核磁共振波谱和x射线晶体学分析了与RIM1 C(2) a结构域密切相关的RIM1 c2a结构域的结构和生化性质。我们发现RIM2的c2a结构域不结合Ca2+。此外，通过核磁共振实验检测到RIM c2a结构域与SNAP-25和synaptotagmin I的C-2结构域的结合很少，这表明RIM c2a结构域尚未确定的相互作用介导了其功能。使用1.4埃分辨率的数据，RIM2 C(2) a域的晶体结构显示出与其他C-2域相似的β -三明治结构，但表现出独特的静电电荷偶极分布，即β -三明治的一边是高正电荷，另一边是高负电荷。CORD7中涉及的突变位点位于结构域底部的位置和序列保守模式表明，与大多数C-2结构域相比，RIM C(2) a结构域可能通过涉及其底面的不依赖于Ca2+的相互作用发挥作用。
RIMs are large proteins that contain two C-2-domains and are localized at presynaptic active zones, where neurotransmitters are released. RIMs play key roles in synaptic vesicle priming and regulation of presynaptic plasticity. A mutation in the RIM1 C(2)A-domain has been implicated in autosomal dominant cone-rod dystrophy (CORD7). The RIM C2A-domain does not contain the full complement of aspartate residues that commonly mediate Ca2+ binding at the top loops of C2-domains, and has been reported to interact with SNAP-25 and synaptotagmin 1, two proteins from the Ca2+-dependent membrane fusion machinery. Here we have used NMR spectroscopy and X-ray crystallography to analyze the structure and biochemical properties of the RIM2 C2A-domain, which is closely related to the RIM1 C(2)A-domain. We find that the RIM2 C2A-domain does not bind Ca2+. Moreover, little binding of the RIM2 C(2)A-domain to SNAP-25 and to the C-2-domains of synaptotagmin I was detected by NMR experiments, suggesting that as yet unidentified interactions of the RIM C2A-domain mediate its function. The crystal structure of the RIM2 C(2)A-domain using data to 1.4 angstrom resolution reveals a beta-sandwich that resembles those observed for other C-2-domains, but exhibits a unique dipolar distribution of electrostatic charges whereby one edge of the beta-sandwich is highly positive and the other edge is highly negative. The location of the mutation site implicated in CORD7 at the bottom of the domain and the pattern of sequence conservation suggest that, in contrast to Most C-2-domains, the RIM C(2)A-domains may function through Ca2+-independent interactions involving their bottom face.