采用X射线衍射解析人鸟嘌呤核苷酸解离刺激因子(RalGDS)Ras结合结构域的空间结构  

Determination of Human Ral Guanine Nucleotide Dissociation Stimulator Ras Binding Domain by X-ray Diffraction

作　　者：黄雪英[1] 雷明[2] 沈阳[3] 张巍[2] 刘艳丽[2] 刘珂[2] 赵浩斌[2] 祁超[2] 

机构地区：[1]华中师范大学化学学院,武汉430079 [2]华中师范大学生命科学学院湖北省遗传调控与整合生物学重点实验室,武汉430079 [3]多伦多大学结构生物学中心

出　　处：《分析化学》2015年第6期893-898,共6页Chinese Journal of Analytical Chemistry

基　　金：国家自然科学基金(Nos.20772040;31300629);教育部中央高校自主项目(No.CCNU14F01006)资助~~

摘　　要：Ras结合结构域(RBD)是鸟嘌呤核苷酸解离刺激因子(Ral GDS)家族成员C-端的高保守区,通过它连接Ras和Ras相关蛋白。利用Red Wings和SGC-1 screens相关的悬滴法设盘结晶,按体积比1∶1加蛋白液到含1∶100胞内蛋白酶Glu-C(w/w)的结晶溶液(2 mol/L(NH4)2SO4,0.2 mol/L Na Ac,0.1 mol/L HEPES,5%MPD,p H 7.5)中,晶体3天长成可组装大小。利用X-射线晶体衍射技术解析了人Ral GDS的Ras结合域(Ral GDS-RBD)的晶体结构,对比鼠和人Ral GDS-RBD,主要是Ras结合区的C-端不同。人Ral GDS-RBD通过Glu838和Glu840在Ral GDS和Ras蛋白间形成氢键,而在同一位点,鼠Ral GDS-RBD通过Asp820和Asp822形成氢键。人Ral GDS-RBD结构中含ββαββαβ-型三维结构的泛素样构象,一个单体的C-端残基与相邻单体的β折叠形成平行βββββ结构。The Ras binding domain （RBD） is a highly conserved domain in the C-terminal region of Ral guanine nucleotide dissociation stimulator （RalGDS） , 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 NaAc, 0.1 mol/L N-hydroxyethyl piperazine ethanesulfonic acid （ HEPES）, 5 % 1,3-methyl-propanediol （MPD） , pH 7.5 ] with 1 ： 100 （w/w） endoproteinase Glu-C. The differences of Ras-interacting regions between the mouse and human RalGDS-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. Glusa8 and Glus40 amino acid residues of human RalGDS-RBD involve in the formation of hydrogen bonds between RalGDS and Ras proteins, whereas at the same positions of mouse RalGDS-RBD, hydrogen bonds form through Asps：o and Asp822. The overall structure of human RalGDS-RBD adopts an ubiquitin-like conformation, which is characterized by a type tertiary structure, and the C-terminal residues of one monomer form parallel β-sheet interaction with the β-strand of another molecule.

关 键 词：鸟嘌呤核苷酸解离刺激因子(Ral GDS) Ras结合结构域(RBD) RAS蛋白 

分 类 号：O657[理学—分析化学] O629.73[理学—化学]