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

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.