Gu Jing, Isozumi Noriyoshi, Gao Bin, Ohki Shinya, Zhu Shunyi
Group of Peptide Biology and Evolution, State Key Laboratory of Integrated Management of Pest Insects and Rodents, Institute of Zoology, Chinese Academy of Sciences, Beijing, China.
Center for Nano Materials and Technology (CNMT), Japan Advanced Institute of Science and Technology (JAIST), Nomi, Ishikawa, Japan.
Front Microbiol. 2022 Dec 1;13:1053078. doi: 10.3389/fmicb.2022.1053078. eCollection 2022.
Mutation-driven evolution of novel function on an old gene has been documented in many development- and adaptive immunity-related genes but is poorly understood in immune effector molecules. Drosomycin-type antifungal peptides (DTAFPs) are a family of defensin-type effectors found in plants and ecdysozoans. Their primitive function was to control fungal infection and then co-opted for fighting against bacterial infection in plants, insects, and nematodes. This provides a model to study the structural and evolutionary mechanisms behind such functional diversification. In the present study, we determined the solution structure of mehamycin, a DTAFP from the Northern root-knot nematode with antibacterial activity and an 18-mer insert, and studied the mutational effect through using a mutant with the insert deleted. Mehamycin adopts an expected cysteine-stabilized α-helix and β-sheet fold in its core scaffold and the inserted region, called single Disulfide Bridge-linked Domain (abbreviated as sDBD), forms an extended loop protruding from the scaffold. The latter folds into an amphipathic architecture stabilized by one disulfide bridge, which likely confers mehamycin a bacterial membrane permeability. Deletion of the sDBD remarkably decreased the ability but accompanying an increase in thermostability, indicative of a structure-function trade-off in the mehamycin evolution. Allosteric analysis revealed an interior interaction between the two domains, which might promote point mutations at some key sites of the core domain and ultimately give rise to the emergence of antibacterial function. Our work may be valuable in guiding protein engineering of mehamycin to improve its activity and stability.
在许多与发育和适应性免疫相关的基因中，已记录了由突变驱动的旧基因上新功能的进化，但在免疫效应分子方面却知之甚少。果蝇霉素型抗真菌肽（DTAFPs）是在植物和蜕皮动物中发现的一类防御素型效应分子。它们最初的功能是控制真菌感染，随后在植物、昆虫和线虫中被用于对抗细菌感染。这为研究这种功能多样化背后的结构和进化机制提供了一个模型。在本研究中，我们确定了来自北方根结线虫的具有抗菌活性且有一个18聚体插入序列的DTAFPs——美哈霉素的溶液结构，并通过使用缺失该插入序列的突变体研究了突变效应。美哈霉素在其核心支架中采用了预期的半胱氨酸稳定的α螺旋和β折叠结构，插入区域称为单二硫键连接结构域（简称为sDBD），形成了一个从支架突出的延伸环。后者折叠成由一个二硫键稳定的两亲性结构，这可能赋予美哈霉素细菌膜通透性。sDBD的缺失显著降低了其抗菌能力，但同时热稳定性增加，这表明美哈霉素进化过程中存在结构-功能权衡。变构分析揭示了两个结构域之间的内部相互作用，这可能促进核心结构域一些关键位点的点突变，最终导致抗菌功能的出现。我们的工作可能对指导美哈霉素的蛋白质工程以提高其活性和稳定性具有重要价值。
