Probing the Dynamic Aspects of AcrB Function through Disulfide Bond Formation

Abstract

The Resistant Nodulation Division (RND) super family member, tripartite complex AcrA-AcrB-TolC efflux pump is one of the major pumps contributing to the multidrug-resistance in Escherichia coli. AcrB is the inner membrane protein of the efflux pump that is responsible for the recognition and binding of compounds before transportation out of the cell. Understanding the dynamics of AcrB regarding the functional rotation of the monomers in a trimeric functional unit in the process of efflux of drugs is the focus of this study. For this purpose, we created 7 disulfide bond pairs in the periplasmic domain of the AcrB using site directed mutagenesis to study the rotation and flexibility of the monomers. These pairs were chosen to be in close proximity to form inter-subunit disulfide bonds. Western blot analysis revealed the formation of disulfide oligomers under non reducing condition and monomers formation under reducing condition. Furthermore, minimum inhibitory concentration assays revealed the difference in antibiotic susceptibility of single and double Cys mutation in which the majority of double Cys mutation have a higher susceptibility to drugs, supporting that disulfide bond formation has facilitate the reduction of efflux activity. This was further confirmed with EtBr accumulation assays utilizing DTT as the reducing agent. In some cases, the activity of the double Cys-mutations was partially or completely restored by DTT, confirming the deleterious effect of the corresponding disulfide bond on protein activity. These findings lead to new insight in conformational changes critical for the activity of the dynamic AcrA-AcrB-TolC efflux pump in E.coli.