Probing the Dynamics of AcrB Through Disulfide Bond Formation
Prasangi Rajapaksha1, Ankit Pandeya1, Yinan Wei1
1Department of Chemistry, University of Kentucky, Lexington, Kentucky 40506, United States.
ACS Omega
|September 9, 2020
Summary
Disulfide bonds in the AcrB protein of the multidrug resistance efflux pump in Escherichia coli revealed the importance of subunit flexibility for function. Restoring flexibility partially restored pump activity, highlighting key dynamics.
Area of Science:
- Microbiology
- Molecular Biology
- Biochemistry
Background:
- The AcrAB-TolC efflux pump is a key factor in multidrug resistance in Escherichia coli.
- AcrB, an inner membrane protein, recognizes and transports compounds out of the cell.
Purpose of the Study:
- To investigate the functional dynamics of AcrB during drug efflux.
- To understand the role of inter-subunit flexibility in AcrB's function.
Main Methods:
- Site-directed mutagenesis to introduce inter-subunit disulfide bonds in AcrB's periplasmic domain.
- Western blot analysis to confirm disulfide bond formation and reduction.
- Minimum inhibitory concentration (MIC) assays and ethidium bromide accumulation assays to evaluate efflux activity.
Main Results:
- Disulfide bond formation resulted in AcrB oligomers, reducible to monomers.
- Mutations affecting flexibility generally increased susceptibility to efflux pump substrates.
- Dithiothreitol (DTT) reduction partially restored activity in some double cysteine mutants, indicating the importance of movement.
Conclusions:
- Inter-subunit flexibility is crucial for the functional dynamics of the AcrAB-TolC efflux pump.
- Disulfide bond formation restricts conformational changes essential for AcrB function.
- These findings offer new insights into the mechanism of multidrug resistance in E. coli.
