Correlation between AcrB trimer association affinity and efflux activity.

The stability of water-soluble proteins and protein oligomers is tuned to match their biological function, the physiological environment, and the regulation mechanism.1−6 Usually, a protein is only marginally stable. This modest stability is thought to be critical for protein function, which usually requires a certain degree of structural flexibility and involves conformational changes. While steady progress has been made in recent years, our understanding of the stability–activity relationship still lags behind in the area of membrane protein research.7 Over the past 2 decades, much insight has been obtained on the causes and consequences of membrane protein oligomerization,7,8 yet a systematic study investigating the correlation between oligomer stability and in vivo activity of a multispan and multidomain helical membrane protein is still missing. Here, we used Escherichia coli AcrB as a model protein to investigate the relationship between its trimer stability and drug efflux activity. AcrB belongs to the resistance-nodulation-cell-division (RND) family of efflux pumps, which are conserved in all Gram-negative bacteria.9−11 RND family efflux pumps play a major role in multidrug resistance in Gram-negative bacteria. AcrB is an obligate trimer that exists and functions exclusively in the trimeric form (Figure ​(Figure11A).12 Trimeric AcrB associates with membrane fusion protein AcrA and outer membrane protein TolC to form a complex that spans the inner membrane, periplasmic space, and outer membrane.13−16 AcrB conducts the inward flow of protons across the inner membrane to drive conformational changes that facilitate substrate efflux. The structure of AcrB was first solved by X-ray crystallography in the asymmetric trimer form in 2006, which supports a conformational cycling model for drug transport.17,18 To date, more than 30 structures of AcrB have been deposited into the Protein Data Bank. However, crystal structures cannot provide insight into how its trimer affinity affects its drug efflux activity. Figure 1 AcrB structure and sequence alignment. (A) Structure of AcrB trimer with the subunit at the back removed for clarity. Positions of the domains discussed in the text are labeled for the side view. Residues lining the interface between TH1 and TH8 are highlighted ... We have previously created several AcrB mutants with modifications on the periplasmic intersubunit interface that displayed different levels of drug efflux activity.19,20 These sites of mutation are located on a protruding loop critical for trimerization. A single amino acid substitution has been shown to almost completely dissociate the trimer structure (AcrBP223G). In this work, we introduced mutations at three sites on the transmembrane intersubunit interface, including both single, double, and triple replacements (Figure ​(Figure1A),1A), aiming at disrupting trimer association without affecting the structure of individual monomers. Structural characterization and stability measurement indicated that these mutations had little impact on monomer structure and stability, whereas they affected the trimer association affinity. We determined their relative trimer affinities and biologically relevant substrate efflux activities. When the efflux activity of the AcrB variants was plotted against their relative trimer stabilities, the requirement of a threshold stability for function was observed.