谷氨酸棒杆菌anti-σ因子CseE及其突变体与σ因子SigE的相互作用分析

Interaction of anti-σ factor CseE and its mutants with σ factor SigE from Corynebacterium glutamicum

摘要:

[目的]谷氨酸棒杆菌是重要的氨基酸生产菌株,本研究针对SigE与ZAS家族蛋白CseE相互作用机制进行探索研究,重点分析CseE突变体影响与SigE结合能力的机制。[方法]本研究选择谷氨酸棒杆菌ATCC 13032来源的SigE和CseE蛋白为研究目标,利用遗传学方法获得过表达的重组谷氨酸棒杆菌,通过RT-qPCR研究SigE调控sigE和cseE的转录情况。同时,利用ITC和His pull-down实验验证ZAS家族的CseE蛋白与Zn²⁺及SigE的结合情况。之后对CseE蛋白进行功能域分析、多序列比对,研究功能域关键氨基酸位点对SigE结合能力的影响。其次对SigE和CseE蛋白进行分子对接和动力学模拟,分析关键氨基酸影响其结合的机制。[结果]谷氨酸棒杆菌SigE调控基因sigE和cseE的转录并且其活性受CseE蛋白控制。CseE蛋白为ZAS家族蛋白,具有Zn²⁺结合能力。CseE_His83A、CseE_cys87A和CseE_cys90A突变体不会影响与SigE的结合能力,而CseE_C87A-C90A和CseE_{His83A-C87A-C90A}突变体与SigE的结合能力略有下降。分子动力学模拟发现SigE-CseE_C87A-C90A和SigE-CseE_{His83A-C87A-C90A}之间的结合能量为-17.23 kcal/mol和-14.06 kcal/mol,分别比未突变体系结合能量降低22.8%及36.9%。[结论]谷氨酸棒杆菌SigE通过聚集RNA聚合酶来调控基因sigE和cseE的表达。CseE蛋白属于ZAS家族,具有Zn²⁺结合能力同时通过与SigE蛋白互作来抑制SigE活性。CseE_C87A-C90A及CseE_{His83A-C87A-C90A}突变体能影响与SigE结合的能力,减弱对SigE活性的控制。本研究产生的三维结构和确定的氨基酸关键位点为后续探索谷氨酸棒杆菌SigE和CseE响应环境压力机制提供了理论基础。

Abstract:

[Objective] Corynebacterium glutamicum is an important amino acid producer.In this study,we explored the mechanisms of interaction between SigE and ZAS family protein CseE,particularly the interaction between CseE mutants and SigE.[Methods] SigE and CseE proteins from C.glutamicum ATCC 13032 were used.The sigE-overexpressing and cseE-overexpressing strains were developed with the genetics method.Through RT-qPCR,we explored the influence of SigE on the transcription of sigE and cseE.At the same time,we tested the binding of CseE proteins to Zn²⁺ and SigE through isothermal titration calorimetry (ITC) and His pull-down experiments,followed by functional domain analysis and multiple sequence alignment of CseE protein to study the effect of key amino acid sites in the domain on the binding ability of SigE.Then,we conducted molecular docking and molecular dynamic simulation of SigE and CseE proteins to analyze the mechanisms of key amino acids affecting their binding.[Results] SigE of ATCC 13032 regulated the transcription of sigE and cseE and its activity was regulated by the CseE protein.CseE,a ZAS family protein,bound to Zn²⁺.CseE_His83A,CseE_Cys87A,and CseE_Cys90A mutantsdid not affect the binding ability to SigE,while the binding ability of CseE_C87A-C90A and CseE_{His83A-C87A-C90A} mutants to SigE decreased slightly.Molecular dynamic simulation showed that the SigE-CseE_C87A-C90A binding energy and SigE-CseE_{His83-C87A-C90A} binding energy were -17.23 kcal/mol and -14.06 kcal/mol,respectively,22.8% and 36.9% lower than the binding energy between SigE and CseE,respectively.[Conclusion]SigE regulates the expression of sigE and cseE by aggregating RNA polymerase,and its protein activity is regulated by CseE.CseE protein belongs to the ZAS family,which binds to Zn²⁺ and inhibits SigE activity by interacting with SigE protein.CseE_C87A-C90A and CseE_{His83A-C87A-C90A} affect the ability to bind to SigE and weaken the control of SigE activity.The three-dimensional structures and the identified key amino acid sites in this study lay a theoretical basis for further exploring the mechanism of SigE and CseE in C.glutamicum in response to environmental stresses.