Multidrug ABC transporters in bacteria.

Multidrug efflux transporters are a plague in the antibiotic resistance mechanisms as they confer the capacity of bacteria to evade most of current therapies. Although these transporters were initially discovered as proton-motive driven pumps, another class of multidrug efflux transporters has emerged in the mid-90s that are powered by ATP hydrolysis. This new class of transporters belongs to one of the largest families of proteins, the ATP-Binding Cassette (ABC) transporters, which are involved in the influx or efflux of a huge variety of molecules. Tremendous progresses have been made in the recent years regarding the functioning mechanism of multidrug efflux ABC transporters, in particular with the accumulation of 3D structures, but many questions remain unsolved. In this review, we will give an overview of our current knowledge on the structure and function of multidrug ABC transporters with an emphasis on bacterial pumps.

Functionality of membrane proteins overexpressed and purified from E. coli is highly dependent upon the strain.

Overexpression of correctly folded membrane proteins is a fundamental prerequisite for functional and structural studies. One of the most commonly used expression systems for the production of membrane proteins is Escherichia coli. While misfolded proteins typically aggregate and form inclusions bodies, membrane proteins that are addressed to the membrane and extractable by detergents are generally assumed to be properly folded. Accordingly, GFP fusion strategy is often used as a fluorescent proxy to monitor their expression and folding quality. Here we investigated the functionality of two different multidrug ABC transporters, the homodimer BmrA from Bacillus subtilis and the heterodimer PatA/PatB from Streptococcus pneumoniae, when produced in several E. coli strains with T7 expression system. Strikingly, while strong expression in the membrane of several strains could be achieved, we observed drastic differences in the functionality of these proteins. Moreover, we observed a general trend in which mild detergents mainly extract the population of active transporters, whereas a harsher detergent like Fos-choline 12 could solubilize transporters irrespective of their functionality. Our results suggest that the amount of T7 RNA polymerase transcripts may indirectly but notably impact the structure and activity of overexpressed membrane proteins, and advise caution when using GFP fusion strategy.

A multidrug ABC transporter with a taste for GTP.

During the evolution of cellular bioenergetics, many protein families have been fashioned to match the availability and replenishment in energy supply. Molecular motors and primary transporters essentially need ATP to function while proteins involved in cell signaling or translation consume GTP. ATP-Binding Cassette (ABC) transporters are one of the largest families of membrane proteins gathering several medically relevant members that are typically powered by ATP hydrolysis. Here, a Streptococcus pneumoniae ABC transporter responsible for fluoroquinolones resistance in clinical settings, PatA/PatB, is shown to challenge this concept. It clearly favors GTP as the energy supply to expel drugs. This preference is correlated to its ability to hydrolyze GTP more efficiently than ATP, as found with PatA/PatB reconstituted in proteoliposomes or nanodiscs. Importantly, the ATP and GTP concentrations are similar in S. pneumoniae supporting the physiological relevance of GTP as the energy source of this bacterial transporter.