neo Trichosanthin及其突变体Y70A neo Trichosanthin的晶体结构研究

天花粉蛋白（Ｔｒｉｃｈｏｓａｎｔｈｉｎ，ＴＣＳ）的一个亚型，ｎｅｏＴｒｉｃｈｏｓａｎｔｈｉｎ（ｎ－ＴＣＳ），及其突变体Ｙ７０Ａｎ－ＴＣＳ被克隆和表达为重组蛋白。用悬滴汽相扩散法得到ｎ－ＴＣＳ和Ｙ７０Ａｎ－ＴＣＳ的晶体。在ＭａｒＲｅｓｅａｒｃｈ面探测器系统上分别收集了０．２０ｎｍ和０．２０５ｎｍ分辨率的Ｘ射线衍射数据。用同晶差值傅立叶法解析了结构。最后晶体学Ｒ因子分别为０．１８３和０．１８４。键长的

CRYSTAL STRUCTURE OF neo TRICHOSANTHIN AND ITS SITE-DIRECTED MUTANT-Y70A neo TRICHOSANTHIN

An isoform of trichosanthin(TCS), neo-trichosanthin(n-TCS), as well as its site-directed mutant-Y70A neo-trichosanthin, has been cloned and expressed as recombinant protein. Crystals of n-TCS and Y70A n-TCS were grown by handing drop vapor diffusion technique. X-ray diffraction data were collected to 0.20 nm and 0.205 nm separately on a Mar Research IP. The structures were determined by the difference Fourier method. The final R factor are 0.183 and 0.185 respectively. The r.m.s. deviations of bond length are 0.0016 nm and 0.0014 nm, the r.m.s. deviations of bond angle are 2.930° and 2.732° respectively, Although there are 11 amino acids difference between n-TCS and TCS, all those residues are not situated in the active pocket. In addition, most of those amino acid replacements are conservative substitutions. In comparison with the structure of TCS, the hydrogen bonds form between the main chains of those 11 residues in n-TCS and their neighboring residues at the same positions of TCS 11 residues within the secondary structures remain unaltered. Therefore the overall structure of n-TCS is very similar to TCS. The crystal of Y70A n-TCS was allowed to soak in buffer containing 5'-adenosine monophosphate (5'AMP) (10g/l) prior to data collection. No electron density corresponding to adenine can be detected around the active pocket. Furthermore, no adenine can be detected after incubation of Y70A n-TCS with 5'AMP. Those results suggest that Tyr70 is crucial to n-TCS and TCS for substrate recognition, binding and perhaps hydrolysis of N-glycoside bond.