Structural studies of perchloric acid soluble protein (PSP) from rat liver.

我们报道了在大鼠肝脏细胞质部分中分离和鉴定了一种新的高氯酸可溶性蛋白（PSP）。PSP为同二聚体，每个亚基由136个氨基酸残基组成，分子量为14,149 Da。它以不同于RNase a的方式抑制兔网织细胞裂解物中无细胞蛋白的合成。最近，在人单核细胞和小鼠肝脏中发现了一种抑制翻译的14kda蛋白。据报道，在PSP的情况下，14-kDa蛋白及其mrna在肝脏和肾脏中优先表达。此外，编码14-kDa蛋白和PSP的互补DNA （cDNA）序列与编码一个新假设的小蛋白家族（YER057c/YJGF）的cDNA序列高度相似，该小蛋白家族目前功能未知。因此，psp样蛋白的序列在原核生物、蓝藻、真菌和真核生物中高度保守。这些蛋白质的高度进化保守性表明，这些蛋白质在细胞调节中起着重要作用。尽管关于psp的信息越来越多，但还没有三维结构的报道。在以下贡献中介绍了PSP的结晶和初步晶体学表征。首先，构建了一种高效的大肠杆菌高氯酸可溶性蛋白（PSP）表达系统。将编码PSP的cDNA插入细菌诱导表达载体pGEX-4T-1中。质粒导入大肠杆菌后经IPTG表达，重组产物经谷胱甘肽- sepharose 4B亲和层析纯化。纯化产物符合预期的nh2末端序列，但其翻译抑制活性比从大鼠肝脏分离的原PSP低10倍。来自大鼠肝脏的PSP已结晶成适合于高分辨率x射线衍射研究的形式。以聚乙二醇（Mr=8000 Da）为沉淀剂，采用悬挂滴法制备了尺寸为0.5 mm的八面体晶体。这些晶体在内部x射线源上衍射到2.44“A”，在ESRE Grenoble使用弯曲的磁体光束线衍射到1.8“A”。由VM值2.12“a”3/Da和自旋函数计算可知，晶体属于立方空间群P213，晶胞参数a=b=c=89.90“a”，每个不对称单元有2个分子。筛选重原子衍生物发现了一种铂化合物，该化合物与蛋白质上的单个位点结合。对重原子衍生物的进一步筛选表明，一个由两个α螺旋包围的β薄片。结构相似度最高的是信号识别颗粒受体ftsz、choris酸突变酶和热休克同源蛋白70 kDa。少

We have reported isolation and characterization of a novel perchloric acid-soluble protein (PSP) in the cytosolic fraction of rat liver. The PSP is a homodimer, each subunit consisting of 136 amino acid residues with a molecular mass of 14,149 Da. It inhibits cell-free protein synthesis in the lysate of rabbit reticulocyte in a different manner than RNase A.Recently, a 14-kDa protein which inhibit translation was characterized from human monocyte and mouse liver. The 14-kDa proteins and their mRNAs were reported to be preferentially expressed in the liver and kidney as in the case of PSP.Furthermore, the sequences of complementary DNA (cDNA) encoding the 14-kDa protein and PSP are highly similar to those of cDNA encoding a new hypothetical family (YER057c/YJGF) of small proteins with presently unknown function. Thus the sequences of PSP-like proteins are highly conserved in prokaryotic, cyanobacteria, fungi and eukaryotes. The high degree of evolutionary conservation of these proteins … More suggests that these proteins play an important role in cellular regulation.Despite the growing body of information gor PSPs, no three-dimensional structure has benn reported. In the following contribution was present the crystallization and preliminary crystallographic characterization of PSP.At first, an efficient E.coli expression system for the production of a perchloric acid-soluble protein (PSP) has been constructed. cDNA encoding PSP was inserted into an inducible bacterial expression vector pGEX-4T-1. After the plasmid introduced into E.coli was expressed by IPTG, the recombinant product was purified by glutathione-Sepharose 4B affinity chromatography. The purified product showed the expected NH2-terminal sequence, but the translation inhibitory activity of this product was 10 times lower compared with that of authentic PSP isolated from rat liver.PSP from rat liver has been crystallized in a form suitable for high resolution X-ray diffraction studies. Octahedral crystals reaching 0.5 mm in size were produced by hangin drop method using polyethylene glycol (Mr=8000 Da) as precipitant. These crystals diffract to 2.44 "A" on an in-house X-ray source and to l.8"A" using a bending, agnet beamline at ESRE Grenoble. The crystals belong to the cubic space group P213 with unit cell parameters a=b=c=89.90"A" and two molecules per asymmetric unit, as indicated from VM value of 2.12"A"3/Da and self-rotation function computation. Screening for heavy-atom derivatives identified a platinum compound that binds to a single site on the protein. Furhter screening for heavy-atom derivatives indeicated a beta sheet flanled by two alpha helice. The structural similarity gave the best hit ftsz (signal recognition particle receptor), chorismate mutase and heat-shock cognate protein 70 kDa. Less