Correlation between AcrB Trimer Association Affinity and Efflux Activity

Abstract

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.

Structure, Assembly, and Function of Tripartite Efflux and Type 1 Secretion Systems in Gram-Negative Bacteria

“…Between the protomers, there is a small contact interface located between TM1 and TM8. 329 The TMDs of the protomers encircle a lipid filled central cavity 279 , 330 ( Figure 5 D). The extensive periplasmic part with a maximum diameter of 100 Å is formed by the periplasmic loops protruding into the periplasm between TM1 and TM2 as well as TM7 and TM8 ( Figure 5 A).…”

How physical forces drive the process of helical membrane protein folding

“…Oligomers seem to be ~4–7 times more stable than individual monomers, with unfolding free energies of ~16–30 kcal/mol (Otzen, 2003; Curnow & Booth, 2007; Barrera et al , 2008; Veerappan et al , 2011). Oligomerization may also be necessary to achieve the full, functional fold (Valiyaveetil et al , 2002; Veerappan et al , 2011; Ye et al , 2014b). The ability to quickly assume a stable, proper fold may avoid aggregation, as all optimal interaction surfaces simultaneously converge together.…”

“…Many membrane proteins function as oligomers (Meng et al , 2009; Clarke & Gulbis, 2012; Reading et al , 2015), with some exhibiting multiple multimeric states (Kahraman & Haselwandter, 2016; Henrich et al , 2017). Further, mutations that disrupt oligomerization, perhaps even without changing monomeric structure, can cause disease (Ng et al , 2012; Ye et al , 2014b). It is thus important to understand how membrane proteins associate into complexes.…”

Peptide-Based Approach to Inhibition of the Multidrug Resistance Efflux Pump AcrB

“…AcrB is known to function as an asymmetric homotrimer in which three AcrB subunits come together to expel antibiotic substrates through a functional rotation mechanism, where each subunit alternates among three separate conformations . Mutagenesis studies have previously identified an integral helix–helix interaction situated within the inner membrane region of the AcrB trimer . While the exact helix binding motif was not elucidated, this interaction is formed between transmembrane helix 1 (TM1) of one subunit and TM8 of a neighboring subunit (Figure ).…”

“…While the exact helix binding motif was not elucidated, this interaction is formed between transmembrane helix 1 (TM1) of one subunit and TM8 of a neighboring subunit (Figure ). A triple mutant of AcrB’s TM8 was shown to disrupt oligomerization and subsequently efflux activity …”