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

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.