Division of Molecular and Structural Biology, Central Drug Research Institute, PO Box 173, Chattar Manzil, Mahatma Gandhi Marg, Lucknow 226001, India.
Int J Parasitol. 2011 Mar;41(3-4):417-27. doi: 10.1016/j.ijpara.2010.11.003. Epub 2010 Dec 14.
Protein translation in the plastid (apicoplast) of Plasmodium spp. is of immense interest as a target for potential anti-malarial drugs. However, the molecular data on apicoplast translation needed for optimisation and development of novel inhibitors is lacking. We report characterisation of two key translation elongation factors in Plasmodium falciparum, apicoplast-encoded elongation factor PfEF-Tu and nuclear-encoded PfEF-Ts. Recombinant PfEF-Tu hydrolysed GTP and interacted with its presumed nuclear-encoded partner PfEF-Ts. The EF-Tu inhibitor kirromycin affected PfEF-Tu activity in vitro, indicating that apicoplast EF-Tu is indeed the target of this drug. The predicted PfEF-Ts leader sequence targeted GFP to the apicoplast, confirming that PfEF-Ts functions in this organelle. Recombinant PfEF-Ts mediated nucleotide exchange on PfEF-Tu and homology modeling of the PfEF-Tu:PfEF-Ts complex revealed PfEF-Ts-induced structural alterations that would expedite GDP release from PfEF-Tu. Our results establish functional interaction between two apicoplast translation factors encoded by genes residing in different cellular compartments and highlight the significance of their sequence/structural differences from bacterial elongation factors in relation to inhibitor activity. These data provide an experimental system to study the effects of novel inhibitors targeting PfEF-Tu and PfEF-Tu.PfEF-Ts interaction. Our finding that apicoplast EF-Tu possesses chaperone-related disulphide reductase activity also provides a rationale for retention of the tufA gene on the plastid genome.
疟原虫质体（顶复体）中的蛋白翻译是一个很有前景的抗疟药物靶点。然而，针对该靶点优化和开发新型抑制剂所需的顶复体翻译分子数据却很缺乏。我们报告了恶性疟原虫中两个关键的翻译延伸因子，质体编码的延伸因子 PfEF-Tu 和核编码的 PfEF-Ts 的特征。重组 PfEF-Tu 能水解 GTP，并与假定的核编码伴侣 PfEF-Ts 相互作用。EF-Tu 抑制剂 kirromycin 影响 PfEF-Tu 的体外活性，表明质体 EF-Tu 确实是该药物的靶点。预测的 PfEF-Ts 前导序列将 GFP 靶向顶复体，证实 PfEF-Ts 在这个细胞器中发挥作用。重组 PfEF-Ts 介导 PfEF-Tu 的核苷酸交换，PfEF-Tu：PfEF-Ts 复合物的同源建模揭示了 PfEF-Ts 诱导的结构改变，这将加速 GDP 从 PfEF-Tu 中释放。我们的结果证实了两个由不同细胞区室编码的质体翻译因子之间的功能相互作用，并强调了它们与细菌延伸因子在序列/结构上的差异与抑制剂活性的关系的重要性。这些数据为研究针对 PfEF-Tu 和 PfEF-Tu：PfEF-Ts 相互作用的新型抑制剂的影响提供了一个实验系统。我们发现顶复体 EF-Tu 具有伴侣相关的二硫键还原酶活性，这也为质体基因组上 tufA 基因的保留提供了依据。
