AcrB trimer stability and efflux activity, insight from mutagenesis studies
Linliang Yu1, Wei Lu, Yinan Wei
1Department of Chemistry, University of Kentucky, Lexington, Kentucky, United States of America.
Plos One
|December 14, 2011
Summary
Mutating a key proline residue in the Escherichia coli AcrB multidrug transporter disrupts trimer stability and reduces drug efflux activity. Restoring trimer stability partially recovers protein function, highlighting proline's role in AcrB assembly.
Area of Science:
- Biochemistry
- Molecular Biology
- Structural Biology
Background:
- The multidrug transporter AcrB in Escherichia coli is an essential homo-trimer.
- Understanding the assembly of membrane protein oligomers like AcrB is crucial.
- Previous work indicated AcrB monomers fold independently before trimerization.
Purpose of the Study:
- To investigate the role of a specific proline residue (P223) in the AcrB trimer structure and function.
- To explore how mutations affecting this residue impact protein activity and assembly.
Main Methods:
- Site-directed mutagenesis of the AcrB gene to replace P223 with other residues.
- Biochemical characterization of purified mutant proteins.
- In vivo trimerization studies.
- Functional assays measuring drug efflux activity.
- Restoration of function experiments using inter-subunit disulfide bonds.
Main Results:
- Mutation of P223 significantly reduced AcrB activity, with P223G being the least active.
- AcrB(P223G) purified as a monomer but formed a trimer in vivo.
- Introducing an inter-subunit disulfide bond partially restored the function of the P223G mutant.
- The P223 residue acts as a wedge stabilizing the AcrB trimer.
Conclusions:
- The protruding loop, stabilized by P223, is critical for AcrB trimer integrity.
- Disruption of this stabilizing "wedge" reduces trimer stability and drug efflux activity.
- Targeting specific residues involved in oligomerization could be a strategy to modulate transporter function.
