Crystallographic identification of Ca2+ and Sr2+ coordination sites in synaptotagmin IC2B domain
Crystallographic identification of Ca2+ and Sr2+ coordination sites in synaptotagmin IC2B domain
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DOI：
10.1110/ps.04832604
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发表时间：
2004-10-01
期刊：
影响因子：
8
通讯作者：
Patel, DJ
中科院分区：
文献类型：
--
作者：
Cheng, Y;Sequeira, SM;Patel, DJ
Synaptotagmin I具有两个串联的Ca 2+结合C-2结构域，其对于中枢神经系统中的快速同步突触传递是必需的。我们已经解决了C2B域的四种晶体结构，其中一种在1.50埃分辨率下为无阳离子形式，两种在1.04埃（两个结合的Ca 2+离子）和1.65埃（三个结合的Ca 2+离子）分辨率下为Ca 2+结合形式，一种在1.18埃分辨率下为Sr 2+结合形式（一个结合的Sr 2+离子）。四个高度保守的天冬氨酸（D303、D309、D363和D365）和两个主链氧（M302：O和Y364：O）的侧链与水分子一起与两个结合的Ca 2+离子（位点1和2）直接接触。在较高的Ca 2+浓度下，N333的侧链旋转并与D309合作以产生第三个Ca 2+配位位点（位点3）。二价阳离子结合位点1和2的C2B结构域先前确定的NMR NOE模式和滴定研究，辅以定点突变分析。晶体和NMR研究之间的一个差异涉及D371，其不参与与晶体结构中任何鉴定的Ca 2+位点的配位，而其与NMR结构中的位点2中的Ca配位。在Sr的存在下，这也是能够触发胞吐，但效率较低，只有一个阳离子结合位点（网站1）被占领的晶体结构。
Synaptotagmin I has two tandem Ca2+-binding C-2 domains, which are essential for fast synchronous synaptic transmission in the central nervous system. We have solved four crystal structures of the C2B domain, one of them in the cation-free form at 1.50 Angstrom resolution, two in the Ca2+-bound form at 1.04 Angstrom (two bound Ca2+ ions) and 1.65 Angstrom (three bound Ca2+ ions) resolution and one in the Sr2+-bound form at 1.18 Angstrom (one bound Sr2+ ion) resolution. The side chains of four highly conserved aspartic acids (D303, D309, D363, and D365) and two main chain oxygens (M302:O and Y364:O), together with water molecules, are in direct contact with two bound Ca2+ ions (sites 1 and 2). At higher Ca2+ concentrations, the side chain of N333 rotates and cooperates with D309 to generate a third Ca2+ coordination site (site 3). Divalent cation binding sites 1 and 2 in the C2B domain were previously identified from NMR NOE patterns and titration studies, supplemented by site-directed mutation analysis. One difference between the crystal and NMR studies involves D371, which is not involved in coordination with any of the identified Ca2+ sites in the crystal structures, while it is coordinated to Ca in site 2 in the NMR structure. In the presence of Sr, which is also capable of triggering exocytosis, but with lower efficiency, only one cation binding site (site 1) was occupied in the crystallographic structure.