Determination of Human Ral Guanine Nucleotide Dissociation Stimulator Ras Binding Domain by X-ray Diffraction
Determination of Human Ral Guanine Nucleotide Dissociation Stimulator Ras Binding Domain by X-ray Diffraction
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X射线衍射法测定人Ral鸟嘌呤核苷酸解离刺激剂Ras结合域
DOI：
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发表时间：
2015
影响因子：
1.2
通讯作者：
Qi Chao
中科院分区：
文献类型：
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作者：
Liu Yan-Li;Liu Ke;Zhao Hao-Bin;Qi Chao
Ras结合域（RBD）是Ral鸟嘌呤核苷酸解离刺激子（Ral GDS）C端区域的高度保守结构域，作为Ras与Ras相关蛋白之间的交联结构域。使用 Red Wings 和 SGC-I 筛选的坐滴原位蛋白水解方法设置结晶。将等体积的蛋白质溶液和储库溶液[2 mol/L(NH4)2SO4,0.混合，得到适合X射线衍射分析的晶体。 2 mol/L 醋酸钠，0。 1 mol/L N-羟乙基哌嗪乙磺酸(HEPES),5% 1,3-甲基丙二醇(MPD),p H 7. 5]与1∶100(w/w)内切蛋白酶Glu-C。通过X射线衍射分析揭示了小鼠和人Ral GDS-RBD之间Ras相互作用区域的差异，结果表明差异主要在Ras相互作用区域的C端。人Ral GDS-RBD的Glu838和Glu840氨基酸残基参与Ral GDS和Ras蛋白之间氢键的形成，而在小鼠Ral GDS-RBD的相同位置，通过Asp820和Asp822形成氢键。人Ral GDS-RBD整体结构采用类泛素构象，其特点是ββαββαβ型三级结构，一个单体的C端残基与另一个分子的β链形成平行的β折叠相互作用。
The Ras binding domain( RBD) is a highly conserved domain in the C-terminal region of Ral guanine nucleotide dissociation stimulator( Ral GDS),which functions as cross-linking domain between Ras and Ras-related proteins. Crystallization was setup using in situ proteolysis method of sitting drops with Red Wings and SGC-I screens. Crystals suitable for X-ray diffraction analysis were obtained by mixing equal volumes of the protein solution and the reservoir solution [2 mol / L( NH4)2SO4,0. 2 mol/L Na Ac,0. 1 mol / L N-hydroxyethyl piperazine ethanesulfonic acid( HEPES),5% 1,3-methyl-propanediol( MPD),p H 7. 5] with 1 ∶ 100( w / w) endoproteinase Glu-C. The differences of Ras-interacting regions between the mouse and human Ral GDS-RBD were revealed by X-ray diffraction analysis and the results showed that the differences were mainly on the C-terminal of Ras-interacting region. Glu838 and Glu840amino acid residues of human Ral GDS-RBD involve in the formation of hydrogen bonds between Ral GDS and Ras proteins,whereas at the same positions of mouse Ral GDS-RBD,hydrogen bonds form through Asp820 and Asp822. The overall structure of human Ral GDS-RBD adopts an ubiquitin-like conformation,which is characterized by a ββαββαβ-type tertiary structure,and the C-terminal residues of one monomer form parallel β-sheet interaction with theβ-strand of another molecule.