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E. coli TolC Multidrug Efflux Pump

Katie Sears '10 and Kristina Buschur '11


I. Introduction

Gram-negative bacteria employ efflux pumps to expel unwanted materials from their cytoplasm. Cells rely on this process for survival. The pump is transient and consists of three proteins when it is complexed. Two inner-membrane proteins make up the translocase complex, which recognizes specific substrates and provides energy for the expulsion step. When the translocase complex is bound to its export substrate, it recruits a third, outer membrane protein known as TolC. The complex is capable of expelling intruders in a single energy-dependent step across both the outer and inner membranes and across the periplasmic space between them. This is possible because TolC stretches between the two membranes, creating a direct path from the cytoplasm to the extracellular space (Koronakis et al., 2000).

TolC has the capacity to interact with various translocase complexes with different export substrates. Possible substrates (each with their own translocase complex) include large proteins like enzymes and toxins, as well as small agents like detergents, solvents, heavy metals, and antibiotics. Expulsion of these materials is vital to the survival of the cell.

The export of antibiotics by efflux pump is particularly significant because it has been shown to contribute to antibiotic resistance. There are two types of antibiotic resistance: drug-specific and multidrug. Drug-specific antibiotic resistance is the type we typically hear about, sometimes caused by the use of antibiotics which place selective pressure on bacteria. This can lead to advantageous mutations and new strains of bacteria, which are resistant to particular substances. In these kinds of cases, mutations encoding efflux systems have been observed on mobile genetic elements like transposons and plasmids. On the other hand, multidrug-resistant bacteria have efflux systems that are encoded chromosomally and expressed constitutively. The endogenous nature of the multidrug efflux proteins indicates that they have not evolved specifically in response to antibiotic pressures (though they might be upregulated in response to these pressures). These complexes have been around for longer periods of time and are thought to have been intended to perform some sort of housekeeping function. Though they may not have evolved for the purpose of antibiotic export, these complexes have proven advantageous for this challenge to cell survival. (Poole, 2007).

The study of multidrug efflux pumps could lead to new treatments for human bacterial infection. Disruption of efflux machinery could be accomplished directly, through interference of gene or protein expression, or by inhibition of pump assembly (Poole, 2007). For example, it has already been shown that loss of the TolC component can result in lower bacterial survival rates (de Cristobal et al., 2006), making this a possible mechanism of antibacterial chemotherapy treatment.

II. Overall Structure

The TolC protein is a trimer of protomers that are each comprised of 428 residues. The overall shape of the protein is that of a long tube, making it well-suited for channeling antibiotics and other harmful molecules out of the bacterial cell. Its long axis is almost 140 Å long, and the cylinder structure has an internal diameter of 35 Å.

Each of the three individual protomers consists of a peptide chain that weaves up and down four times, forming three distinct domains along the long axis of the molecule: a β-barrel domain, an α-helical domain, and a mixed α/β equatorial domain. The β-barrel domain consists of four β-sheets which appear at the distal (upper) end of the structure. The α-helical domain is made up of 6 α-helices, two of which extend to the proximal (lower) end of the structure. The equatorial domain is two small β-sheets and three small α-helices that form a band around the middle of the helical barrel. A topology diagram of the protomer shows the secondary structure elements, their position relative to one another, and their location in the overall structure.

In the trimer structure, the antiparallel β-sheets form a 12-stranded, right twisted β-barrel. The barrel is approximately 40 Å long. In the cell, the β-barrel end of the molecule faces the outer membrane. The α-helices form a 12-stranded antiparallel, left-twisted barrel with an internal diameter of 35 Å and length 100 Å. The internal space has a volume of approximately 43,000 Å3; it’s one of the largest ever discovered in a protein. In the cell, the α-helical barrel extends into the periplasm. At the distal end the structure is open, but at the proximal end it tapers almost to a close. Where the β-barrel and the α-helical barrel meet, abrupt turns in proline-containing interdomain linkers accommodate the transition from a left-twisted barrel to a right-twisted barrel. (Koronakis et al., 2000)

III. β-Barrel Domain

Unlike other outer membrane factors (OMFs) that have been characterized, the TolC OMF β-barrel is wide open and accessible to molecules such as solvent. In order to maintain this conformation, the β strands must be twisted and curved. This is accomplished by the placement of small or unbranched residues—including serine and aspartic acid, which can pack closely with other molecules—toward the inside of the barrel. In addition to giving the barrel its shape, these small side chains contribute to the open conformation of the barrel. In contrast, larger, more bulky residues have side chains outside of the barrel. In particular, the aromatic residues phenylalanine and tyrosine form clusters on the outside of the barrel that define where TolC OMF meets the inner edge of the outer E. coli outer membrane. This is a clear indication that the TolC protein is positioned similarly to other OMFs that have been previously characterized (Koronakis et al., 2000).

IV. α-Helical Domain

The α-helical barrel extends into the periplasm of the bacterial cell. The helices curve and twist around each other and are considered coiled coils. They have a left-handed superhelical twist throughout the entire barrel, but untwist somewhat near the distal end of the barrel. The two long helices from each protomer (H3 and H7) remain uniform from the distal end of the barrel to where the equatorial domain curves around the barrel. From that point till they reach the proximal end of the barrel they begin to gradually taper in. Helices H7 and H8 form a conventional coiled coil in which both helices twist. On the other hand, H3 remains straight while H4 coils around it. (Koronakis et al., 2000)

The coiled coils also interact with each other. For example, Asp153 on H4 hydrogen bonds with Tyr362 on H7 and Gln136 on H3 hydrogen bonds with Glu359 on H7 (Andersen et al., 2002). Many residues, such as these, that participate in hydrogen bonds between the coiled coils are highly conserved in the TolC family.

V. Transport Mechanism

TolC interacts with several inner membrane translocase complexes to be able to export a large variety of materials from the cytoplasm. To export molecules as large as whole proteins, the tapered proximal end of TolC must be able to open and the proteins to be exported must be at least partially unfolded. It is possible that this opening happens when the inner H7/H8 coiled coils rotate slightly around the outer H3/H4 coiled coils. Koronakis et al. propose that this small change in the structure of TolC could open the tunnel by as much as 30 Å (2000). They also suggest that this change is induced by the recruitment of TolC by a translocase complex. It’s likely that the equatorial domain recognizes the translocase complex interaction because its β-sheets and α-helices are arranged close to the inner H7/H8 set of coiled coils and any change in their relative positions could induce uncoiling of the helices.

TolC is recruited by a translocase that has bound a particular substrate that is to be exported. This provides specificity for what is exported and allowed access to the TolC tunnel. When TolC is complexed with the translocase, a transient connection directly from the cytoplasm, across the inner and outer membranes and periplasm, and to the outside of the cell is established. There is a high degree of sequence similarity among members of the TolC family, suggesting that this proposed mechanism for transport could be common to all bacterial efflux pumps. (Koronakis et al., 2000)

VI. Export Complex

TolC is the outer membrane protein of many multidrug efflux pumps. The major drug efflux pump of E. coli is the AcrA-AcrB-TolC complex.This transport protein complex is part of the resistance nodulation cell division (RND) family—one of five major classes of bacterial efflux systems (Poole, 2007).

AcrA is a periplasmic protein in the efflux pump complex. Here, the stable core of AcrA was crystallized with four molecules per asymmetric unit. One AcrA protomer looks like this: . The AcrA molecule is thought to be trimeric (a complex of three of these protomers) in vivo, but this has not been proven. Each protomer of this protein has three domains: a β-barrel, a lipoyl domain, and a coiled-coil α-helical domain. AcrA belongs to the membrane fusion protein (MFP) family, which includes proteins that also play a role in two other efflux pump families: ATP-binding cassette (ABC) and major facilitator system (MFS). Though it is not known precisely how AcrA interacts with TolC and the third protein of the efflux pump—AcrB—Mikolosko et al. postulate that, in addition to helping maintain the formation of the efflux pump complex, conformational changes in AcrA might also play a role in TolC channel opening (2006).

AcrB spans the inner membrane. It is a trimer consisting of three molecules that create a small channel. These three molecules cycle through three different conformations in order to propel substrate molecules from the cytoplasm toward the TolC OMF, to be expelled from the cell. The conformational changes that make this molecule work are fueled by proton-motive force within the transmembrane domain. One end of the AcrB molecule, oriented toward the cytoplasm, serves as an occlusion site where substrate molecules enter the pump. This domain is made up mostly of α-helicies. The other end of the molecule, located in the periplasm, acts as the TolC docking domain and is a complex structure of α-helicies and β-sheets. The docking domain is where the α-helical channel of TolC fits into the AcrB inner membrane protein. Little is known about the specific interactions between the two proteins at this time (Seeger et al., 2006).

VII. References

Andersen, C, Koronakis, E, Bokma, E, Eswaran, J, Humphreys, D, et al. Transition to the open state of the TolC periplasmic tunnel entrance. Proc Natl Acad Sci U S A. 2002. 99:11103–11108.

De Cristobal, R.E., Vincent, P.A., Salomon, R.A. Multidrug resistance pump AcrAB-TolC is required for high-level, Tet(A)-mediated tetracycline resistance in Escherichia coli. Journal of Antimicrobial Chemotherapy. 2006. 58:31-36.

Koronakis, V., Sharff, A., Koronakis, E., Luisi, B., Hughes, C. Crystal structure of the bacterial membrane protein TolC central to multidrug efflux and protein export. Nature. 2000. 405:914-919.

Mikolosko, J., Bobyk, K., Zgurskaya, H.I., Ghosh, P. Conformational Flexibility in the Multidrug Efflux System Protein AcrA. Structure. 2006. 14:577-587.

Poole, K. Efflux pumps as antimicrobial resistance mechanisms. Annals of Medicine. 2007. 39:162-176.

Seeger, M.A., Schiefner, A., Eicher, T., Verrey, F., Diederichs, K., Pos, K.M. Structural Asymmetry of AcrB Trimer Suggests a Peristaltic Pump Mechanism. Science. 2006. 313:1295-1298.

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