Correlation between AcrB trimer association affinity and efflux activity.
The majority of membrane proteins function as oligomers. However, it remains largely unclear how the oligomer stability of protein complexes correlates with their function. Understanding the relationship between oligomer stability and activity is essential to protein research and to virtually all cellular processes that depend on the function of protein complexes. Proteins make lasting or transient interactions as they perform their functions. Obligate oligomeric proteins exist and function exclusively at a specific oligomeric state. Although oligomerization is clearly critical for such proteins to function, a direct correlation between oligomer affinity and biological activity has not yet been reported. Here, we used an obligate trimeric membrane transporter protein, AcrB, as a model to investigate the correlation between its relative trimer affinity and efflux activity. AcrB is a component of the major multidrug efflux system in Escherichia coli. We created six AcrB constructs with mutations at the transmembrane intersubunit interface, and we determined their activities using both a drug susceptibility assay and an ethidium bromide accumulation assay. The relative trimer affinities of these mutants in detergent micelles were obtained using blue native polyacrylamide gel electrophoresis. A correlation between the relative trimer affinity and substrate efflux activity was observed, in which a threshold trimer stability was required to maintain efflux activity. The trimer affinity of the wild-type protein was approximately 3 kcal/mol more stable than the threshold value. Once the threshold was reached, an additional increase of stability in the range observed had no observable effect on protein activity.
Department of Chemistry, University of Kentucky , Lexington, Kentucky 40506, United States.
Cui
C
Ye
Zhaoshuai
Z
Wang
Wei
W
Lu
Meng
M
Zhong
Qian
Q
Chai
Yinan
Y
Wei
D019943:Amino Acid Substitution; D000900:Anti-Bacterial Agents / Q000378:metabolism* / Q000494:pharmacology; D001692:Biological Transport; D024901:Drug Resistance, Multiple, Bacterial; D004917:Erythromycin / Q000378:metabolism* / Q000494:pharmacology; D004926:Escherichia coli / Q000187:drug effects / Q000254:growth & development / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004996:Ethidium / Q000378:metabolism; D005456:Fluorescent Dyes / Q000378:metabolism; D007700:Kinetics; D008826:Microbial Sensitivity Tests; D008958:Models, Molecular; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D050505:Mutant Proteins / Q000378:metabolism; D009675:Novobiocin / Q000378:metabolism* / Q000494:pharmacology; D009861:Onium Compounds / Q000378:metabolism; D009943:Organophosphorus Compounds / Q000378:metabolism; D055550:Protein Stability; D020836:Protein Structure, Quaternary; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012235:Rhodamines / Q000378:metabolism
D000900:Anti-Bacterial Agents; D004917:Erythromycin; D029968:Escherichia coli Proteins; D004996:Ethidium; D005456:Fluorescent Dyes; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D050505:Mutant Proteins; D009675:Novobiocin; D009861:Onium Compounds; D009943:Organophosphorus Compounds; D021122:Protein Subunits; D011994:Recombinant Proteins; D012235:Rhodamines; C083496:AcrB protein, E coli; C026188:rhodamine 6G; C013289:tetraphenylphosphonium