Wawiórka Leszek, Krokowski Dawid, Gordiyenko Yuliya, Krowarsch Daniel, Robinson Carol V, Adam Ishag, Grankowski Nikodem, Tchórzewski Marek
Department of Molecular Biology, Maria Curie-Skłodowska University, Akademicka 19, 20-033 Lublin, Poland.
Department of Chemistry, University of Oxford, South Parks Rd, Oxford OX1 3QZ, UK.
Biochim Biophys Acta. 2015 Jan;1850(1):150-8. doi: 10.1016/j.bbagen.2014.10.015. Epub 2014 Oct 23.
The ribosomal stalk composed of P-proteins constitutes a structure on the large ribosomal particle responsible for recruitment of translation factors and stimulation of factor-dependent GTP hydrolysis during translation. The main components of the stalk are P-proteins, which form a pentamer. Despite the conserved basic function of the stalk, the P-proteins do not form a uniform entity, displaying heterogeneity in the primary structure across the eukaryotic lineage. The P-proteins from protozoan parasites are among the most evolutionarily divergent stalk proteins.
We have assembled P-stalk complex of Plasmodium falciparum in vivo in bacterial system using tricistronic expression cassette and provided its characteristics by biochemical and biophysical methods.
All three individual P-proteins, namely uL10/P0, P1 and P2, are indispensable for acquisition of a stable structure of the P stalk complex and the pentameric uL10/P0-(P1-P2)₂form represents the most favorable architecture for parasite P-proteins.
The formation of P. falciparum P-stalk is driven by trilateral interaction between individual elements which represents unique mode of assembling, without stable P1-P2 heterodimeric intermediate.
On the basis of our mass-spectrometry analysis supported by the bacterial two-hybrid assay and biophysical analyses, a unique pathway of the parasite stalk assembling has been proposed. We suggest that the absence of P1/P2 heterodimer, and the formation of a stable pentamer in the presence of all three proteins, indicate a one-step formation to be the main pathway for the vital ribosomal stalk assembly, whereas the P2 homo-oligomer may represent an off-pathway product with physiologically important nonribosomal role.
由P蛋白组成的核糖体柄是大核糖体颗粒上的一种结构，负责在翻译过程中招募翻译因子并刺激因子依赖性GTP水解。柄的主要成分是P蛋白，它们形成一个五聚体。尽管柄具有保守的基本功能，但P蛋白并不形成一个统一的实体，在真核生物谱系中的一级结构上表现出异质性。原生动物寄生虫的P蛋白是进化上分歧最大的柄蛋白之一。
我们利用三顺反子表达盒在细菌系统中体内组装了恶性疟原虫的P柄复合物，并通过生化和生物物理方法对其特性进行了研究。
所有三种单独的P蛋白，即uL10/P0、P1和P2，对于获得P柄复合物的稳定结构都是不可或缺的，五聚体uL10/P0-(P1-P2)₂形式代表了寄生虫P蛋白最有利的结构。
恶性疟原虫P柄的形成是由各个元件之间的三边相互作用驱动的，这代表了一种独特的组装模式，没有稳定的P1-P2异二聚体中间体。
基于我们的质谱分析，并得到细菌双杂交试验和生物物理分析的支持，我们提出了寄生虫柄组装的独特途径。我们认为，不存在P1/P2异二聚体，以及在所有三种蛋白质存在的情况下形成稳定的五聚体，表明一步形成是重要核糖体柄组装的主要途径，而P2同寡聚体可能代表一种具有重要生理非核糖体作用的非主要途径产物。
