关于N-degron途径在蛋白质降解网络中的重要性,衔接蛋白ClpS识别带有经典N-degrons的底物,并将它们输送到酪蛋白分解蛋白酶复合物ClpAP中进行降解。有趣的是,位于蛋白质底物N端附近的大多数N-degrons属于疏水型氨基酸。叶绿体,植物光合作用的重要细胞器,包含多样化的Clp降解系统。尽管有几项研究证实,叶绿体ClpS能够与源自原核生物的经典N-degrons相互作用,然而,迄今为止,叶绿体ClpS蛋白如何识别N-degrons标记的底物的分子机制尚不清楚。莱茵衣藻是一种用于光合作用研究的单细胞模型生物,拥有一个大的杯状叶绿体,相应的基因组数据表明它拥有细菌同源衔接蛋白,命名为CrClpS1。然而,相关的生化知识,CrClpS1接头的蛋白质结构研究迄今尚无报道。CrClpS1与可能的N-degrons之间的分子相互作用也未定义。这里,我们建立了可靠的CrClpS1同源模型,并找到了N-degron结合的疏水口袋。我们结合了分子对接,分子动力学模拟,和MM/PBSA,MM/GBSA结合自由能估计,以阐明CrClpS1-N-degron相互作用的分子性质。此外,我们研究了CrClpS1-apo在水-溶剂环境中的构象变化,并通过长时间的分子动力学模拟分析了其可能的生物学意义。具体来说,适配器CrClpS1显示与Phe更强的相互作用,Trp,Tyr,His和Ile相对于其他氨基酸。使用残留物分解分析,CrClpS1和N-degrons之间的相互作用很大程度上取决于几个保守残基,它们位于疏水口袋周围,暗示从莱茵衣藻中分离出的叶绿体采用相对保守的N-degron识别模式。此外,CrClpS1疏水口袋的开合可能有利于N-degron选择性。
Regarding the importance of N-degron pathway in protein degradation network, the adaptor protein ClpS recognizes the substrates bearing classical N-degrons, and delivers them to caseinolytic protease complex ClpAP for degradation. Interestingly, the majority of N-degrons located near the N-terminus of protein substrate are belonged to the hydrophobic type amino acids. Chloroplast, an important organelle for plant photosynthesis, contain a diversified Clp degradation system. Despite several studies have confirmed that chloroplastic ClpS is able to interact with classical N-degrons derived from prokaryotes, whereas, the molecular mechanism underlying how the chloroplastic ClpS protein could recognize the substrate tagged by N-degrons is still unclear until now. Chlamydomonas reinhardtii is a kind of unicellular model organism for photosynthesis researches, which possesses a large cup-shaped chloroplast, and the corresponding genome data indicates that it owns bacterial homologous adaptor protein, named CrClpS1. However, the relevant biochemical knowledges, and protein structure researches for CrClpS1 adaptor aren\'t reported up to date. The molecular interactions between CrClpS1 and possible N-degrons are undefined as well. Here, we build a reliable homology model of CrClpS1 and find a hydrophobic pocket for N-degron binding. We combine molecular docking, molecular dynamic simulations, and MM/PBSA, MM/GBSA binding free energy estimations to elucidate the molecular properties of CrClpS1-N-degron interactions. Besides, we investigate the conformational changes for CrClpS1-apo in water-solvent environment and analyze its possible biological significances through a long time molecular dynamic simulation. Specifically, the adaptor CrClpS1 displays the stronger interactions with Phe, Trp, Tyr, His and Ile with respect to other amino acids. Using the residue decomposition analysis, the interactions between CrClpS1 and N-degrons are heavily depended on several conservative residues, which are located around the hydrophobic pocket, implying that chloroplast isolated from Chlamydomonas reinhadtii adopts a relatively conservative N-degron recognition mode. Besides, the opening-closure of hydrophobic pocket of CrClpS1 might be beneficial for the N-degron selectivity.