基于结构计算设计提高费氏弧菌LuxR蛋白转录激活能力的策略  

Enhancing the Transcription Activation Capability of LuxR Protein in Vibrio Fischeri through Structure-based Computational Design

作　　者：徐依聪 李家璜 华子春 XU Yicong;LI Jiahuang;HUA Zichun(School of Biopharmacy,China Pharmaceutical University,Nanjing 211198,China;The State Key Laboratory of Pharmaceutical Biotechnology,School of Life Sciences,Nanjing University,Nanjing 210023,China;Changzhou High-Tech Research Institute of Nanjing University and Jiangsu Target Pharma Laboratories Inc.,Changzhou 213164,China;Jiangsu Society of Biochemistry and Molecular Biology,Nanjing 210019,China;Nanjing Genrecom Laboratoreis Ltd.,Nanjing 211899,China)

机构地区：[1]中国药科大学生物药物学院,江苏南京211198 [2]南京大学生命科学学院,医药生物技术国家重点实验室,江苏南京210033 [3]常州南京大学高新技术研究院,江苏省产业技术研究院医药生物技术研究所,江苏常州213164 [4]江苏省生物化学与分子生物学学会,江苏南京210019 [5]南京吉芮康生物科技研究院,江苏南京211899

出　　处：《药物生物技术》2024年第3期221-227,共7页Pharmaceutical Biotechnology

基　　金：国家自然科学基金资助项目(No.82130106,No.32250016,No.82303774);江苏省科技厅基础研究计划自然科学基金项目(No.BK20230165,No.BE2023695);常州市科技计划项目(No.CJ20230017,No.2022169)资助。

摘　　要：采用理性设计策略提高费氏弧菌转录因子LuxR蛋白的转录激活能力。利用蛋白质建模和分子对接方法构建了LuxR同源二聚体-3-oxo-C6-HSL复合物,进一步采用分子动力学模拟并进行结合自由能分析,寻找影响LuxR活性的潜在突变位点,最终借助虚拟饱和突变筛选有利突变体,并通过构建LuxR突变质粒验证突变体的转录激活能力。研究结果显示,Ser54对LuxR二聚化有重要影响,而虚拟饱和突变提示S54L、S54F、S54W与S54Y 4个突变能够提升LuxR二聚体的稳定性,这可能有助于提升其转录激活能力;实验验证发现当3-oxo-C6-HSL的浓度为10^(-5)mol/L时,S54W和S54L能够将LuxR的活性提高至WT的1.4倍。成功利用计算机辅助设计技术筛选出高活性LuxR突变体,为LuxR蛋白家族以及其他二聚体蛋白的改造提供新思路。Rational design strategy was employed to enhance the transcriptional activation capability of the LuxR protein from Vibrio fischeri.In this study,Alpha Fold-multimer was utilized for modeling LuxR homodimers.AutoDock Vina was employed to rigidly dock 3-oxo-C6-HSL into the ligand binding pockets located at the N-terminus of each subunit of the dimer,resulting in the LuxR homodimer-3-oxo-C6-HSL complex.Furthermore,Gromacs was used to perform 100 ns molecular dynamics simulations on the obtained models.The binding free energy and amino acid energy decomposition between dimers in the simulated results were calculated using the MM-PBSA method,aiming to identify potential mutation sites that could affect LuxR activity.Upon determining the mutation site,virtual amino acid saturation mutagenesis was conducted at the site using MOE and Discovery studio software to compute the potential effects of mutations.To validate the results of virtual screening,fluorescent protein assays were conducted to compare the transcriptional activation capabilities of mutant variants.The analysis of RMSD and Rg suggested that the LuxR homodimer-3-oxo-C6-HSL complex maintained stability throughout the simulation.Additionally,secondary structure analysis result also showed no significant changes in the internal conformation of the protein.Energy decomposition results of inter-dimer MM-PBSA binding free energy suggested that Ser54 had a significant impact on LuxR dimerization.Subsequent virtual saturation mutagenesis at this site revealed that the S54L,S54F,S54W,and S54Y mutations may contribute to the stability of the LuxR dimer,potentially enhancing its transcriptional activation capability.The fluorescent protein assays demonstrated that when the concentration of 3-oxo-C6-HSL in the solution was 10^(-5) mol/L,the S54W and S54L mutations could enhance LuxR activity to 1.4 times that of the wild type.This study successfully utilized protein modeling,molecular dynamics simulations,and virtual saturation mutagenesis among other computeraided des

关 键 词：LuxR蛋白 理性设计 分子动力学模拟 结合自由能 虚拟饱和突变 定点突变 

分 类 号：Q81[生物学—生物工程]