TolC: a microscopic hoover for antibiotics

By Francesco Rivetti

Unfortunately for humanity, bacteria have evolved proteins, called TolC, that enable them to expel any hydrophobic substances, essentially acting like microscopic antibiotic-hoovers. Such structures are one of the reasons why multi-resistant bacteria are evolving, as this protein provides protection from a wide array of drugs. In 1981, Morona and Reeves noticed that E. coli with a mutation to a small transmembrane protein – TolCshowed remarkable resistance to some bacteriocins (Zgurskaya et al., 2019), which are bacterial synthesized toxins that inhibit the growth of related bacteria strains (, 2019). Later studies, e.g. Brenz R. (1993), confirmed that TolC was involved in the export of toxins out of bacterial cells. From that point forward, interest in TolC grew rapidly as its potential was uncovered: the genesis of multi-drug resistant bacteria (Zgurskaya et al., 2019). 

TolC is a 140 kD protein found in Gram-Negative bacteria, hence the pore forms complexes in order to span both membranes (Buchanan, 2019). TolC is a heterotrimer composed of a 140-Å long 12 stranded alpha/beta barrel (Koronakis V, 2019). The transmembrane domain is composed of three identical four right-handed antiparallel beta-sheets (10-13 aa) that form a beta-barrel connected by loops (, 2019). The alpha-helical domain instead extends through the periplasm and it is formed by a set of three alpha-helices, containing six polar R-groups (, 2019). Whereas the equatorial domain is composed of three sets of two beta-sheets and two alpha-sheets. The alpha-helical domain is separated into two sections (see diagram I). The first section is composed of an alpha-barrel, whose sidechains that extend inside are smaller and the ones on the outside instead are larger and bulkier: enforcing an alpha-barrel arrangement (, 2019). Instead, the second lower section is arranged in coiled coils, which diminish in width towards the end to seal the channel (, 2019). This is done to prevent leaking or entering of molecules that are larger than small ions or water (, 2019). The coiled-coil section is formed by six pairs of alpha-helices, which pack together due to the hydrophobic effect as they contain similar-sized non-polar R-groups (, 2019). The narrowness of the coiled coils is maximized by a set of polar and charged side chains at the very end of the helices, the protein contains six hydrogen bonds making the tube as narrow as 5Å (, 2019). The importance of these H-bonds was highlighted with a mutated form of TolC: Arg367 to Ser and Tyr362 to Phe, where the breaking of a single H-bond increased the radius by 1.5Å: increasing ion leakage (, 2019). 

In order to span the two membranes, TolC forms a trimeric complex, composed of two ArcA molecules and one AcrB and TolC (see diagram II). AcrA, also known as the adaptor protein, is composed of four domains, one of which extends into the inner PM of the bacterium via an N-terminal lipid-anchor (Seeger et al., 2008). AcrA binds to the two lateral sides of AcrB and TolC, it is hypothesized that it aids the other proteins to undergo the required conformational changes for the complex-formation. AcrB instead is a 3’096 residue-long homotrimer composed of a transmembrane, pore and TolC-binding domain (Seeger et al., 2008). The protein has a substrate binding-site and provides the energy by coupling it with H+ antiport: where protons diffuse out of the periplasm down their concentration gradient (Müller and Poos, 2019). The complex is yet to be completely understood but from the latest literature it is known that the adaptor – AcrA –  binds with its alpha-harpin domain to the alpha-helical domain of TolC, whereas the AcrA/B interactions are not understood in detail (Seeger et al., 2008).     

TolC is involved in a Type I secretory pathway, consisting of three components, all of which can be cross-linked to form a continuous pore-complex (Buchanan, 2019). When the substrate-complex in the inner membrane forms it recruits and binds to TolC, causing the constrictions at the end of the alpha-helix domain of TolC to loosen (Andersen et al., 2019). Thus, enabling the passage of larger substrates (Andersen et al., 2019). The actual mechanism of this opening has not been cracked yet; nevertheless, possible mechanisms have been proposed. The most promising seems to be an allosteric one, in which complex formation causes the realignment of the coiled-coils with the overlying alpha-barrel: increasing the diameter of the lower alpha-helical lumen (Zgurskaya et al., 2019). A hypothetical realigned open-state was developed, and its structure was compared to the closed-state: elucidating a possible mechanism. The answer seems to lie in the previously mentioned hydrogen bonds, as the hypothetical open state lacked any of them. Inter- and inner-chain hydrogen-bonds and salt-bridges at the end of the alpha-helices were destroyed. The mechanism predicted that the inner coiled-coil alpha-helices (H7/8) would realign with the outer coils (H3/4) (Zgurskaya et al., 2019). Therefore, the cumulative breaking of the non-covalent interactions appears to be enough to cause the lower part of the alpha-helices to realign and form a continuous alpha-barrel, lacking coiled coils causing a conformational change from a tapered structure to a more cylindrical one (Zgurskaya et al., 2019). Further studies have supported this by studying mutant TolC proteins, which had disrupted hydrogen bonds and therefore did not function properly. 

The mechanism of substrate-efflux is quite well understood. First, the substrates will be either by or in the PM on the periplasmic side, due to their hydrophobic nature (Seeger et al., 2008). Therefore, a monomer will bind to the non-polar substrate and it will be brought to the hydrophobic binding-pocket on AcrB, which is composed of many non-polar residues (Seeger et al., 2008). Secondly, the substrate-binding causes a conformational change, which moves the binding-site to the distal end of the vestibule, close to the top of the pore domain (, 2019). Lastly, a further conformational change occurs, where the hydrophobic binding-site is hidden, and an aperture opens close to the AcrB-TolC attachment. Therefore, the substrate is extruded through the efflux pore (, 2019). The transformation from the second to the third step is speculated to be the most energy intensive (Seeger et al., 2008). Therefore, it is thought that a proton from the periplasm binds to AcrB during the second step and it is then released in the cytoplasm during the third step: enforcing the required conformational changes in the complex (Seeger et al., 2008). To summarize, the hydrophobic toxin binds to the hydrophobic pocket, which then is moved towards the outer membrane and then hidden, forcing the toxin out of the bacterial cell.

This protein is any microbiologist’s nightmare: a promiscuous hydrophobic antibiotic hoover. Because of this danger, researchers have focused on figuring out how to prevent the pump’s formation. Its relevance in virality was highlighted when its inactivation was found to increase the susceptibility of bacteria to various types of antimicrobials (Sulavik et al., 2019) and cause metabolic shutdowns and growth defects. Unfortunately, the pumps function does not stop at antibiotic excretion, as studies have linked the protein with low-pH resistance, which is due to its proton antiport function. This has been linked to the increase in antibiotic-susceptibility at high pHs, where proton transport in the cytosol becomes less favourable. Efflux Pump Inhibitors (EPIs) have an extreme potential as their inactivation would increase bacterial antibiotic-sensitivity, (Abdali et al., 2019). A recent study found four molecules that bind to AcrA, causing complex formation inhibiting conformational changes. Two of these molecules, clorobiocin and SLU-258, are thought to likely bind to the center of AcrA, between the lipoyd and beta-barrel domains: therefore, acting as possible drugs that could restore other antibiotics’ function (Darzynkiewicz et al., 2019).  


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