【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。更多还原

【Abstract】 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.更多还原