Structure of the MacAB–TolC ABC-type tripartite multidrug efflux pump

The MacA–MacB–TolC assembly of Escherichia coli is a transmembrane machine that spans the cell envelope and actively extrudes substrates, including macrolide antibiotics and polypeptide virulence factors. These transport processes are energized by the ATPase MacB, a member of the ATP-binding cassette (ABC) superfamily. We present an electron cryo-microscopy structure of the ABC-type tripartite assembly at near-atomic resolution.
Structure of the MacAB–TolC ABC-type tripartite multidrug efflux pump

Multi-drug resistance is one of the three greatest risks to human health according to the WHO. It is broadly defined as the resistance of pathogens or even cancer cells against multiple drugs – whether administered one following another, or simultaneously as a drug cocktail. Antibiotic resistance claims around 25,000 lives per annum in Europe alone, a fatality rate equivalent to a catastrophic jumbo jet crash every week over the course of a full year.

One of the major mechanisms of multiple antibiotic resistance in bacteria are multi-drug efflux pumps. These membrane proteins recognize, bind, and expel a variety of toxic substrates, including antibiotic drugs, on diffuse through the membrane system into the cell interior. Structure determination of these pumps lays the foundation for a better understanding of how these molecular vacuum cleaners work and guide the development of inhibitors and novel types of antibiotics.

We have set ourselves the aim to use cryo-electron microscopy to solve one of the most important yet outright challenging gram-negative multi-drug efflux systems – the macrolide antibiotics exporting system MacB-MacA-TolC, a member of the ATP-binding cassette multi-drug efflux pump family.

In the MacAB-TolC multi-component efflux system, the efflux of macrolide antibiotics (and various other substrates) is energized from the adenosine-triphosphate hydrolysis by MacB in the inner membrane. A hexamer of MacA, connecting MacB with the trimeric outer membrane channel TolC, establishes a pathway for substrate efflux. Nature has found a beautiful synchronisation of these three versatile players to coordinate efficient complex assembly: First, an interaction of MacA and MacB is stimulated by the presence of ATP as the energy source for efflux. Secondly, this assembly recruits TolC into the full and active pump complex.

Whereas structures of the individual components MacA and TolC were previously solved, the structure of MacB and the full complex assembly, including the complex’s stoichiometry, have remained a complete mystery and were chased by generations of adventurous structural biologists. However, this quest has finally achieved its goal.

One of the main challenges in obtaining a high-resolution of the full ca. 550 kDa-large system was the purification of the whole complex in sufficient quantity and quality for single particle analysis. The quality of the latter defines the quality of the averaged final result.

Using a combination of MacA-MacB-fusion and -cysteine cross-linking in our construct design, we finally solved the full map of a functional MacB-MacA-TolC complex from E. coli using cryo-EM. The complex revealed a rather unusual stoichiometry of 2:6:3 (MacB:MacA:TolC). Exploring the map in greater detail, we observe an interesting constriction point in the MacA-hexamer which is formed by a conserved glutamine ring. This glutamine ring may act as a ‘one-way-only’ gate through which substrates passively move without the possibility of backflow, e.g. in situations where the efflux pump operates against a concentration gradient.

The detailed map of the full complex now opens new exciting possibilities for in-depth functional analysis in the future.

Read the full paper in Nature Microbiology: Structure of the MacAB–TolC ABC-type tripartite multidrug efflux pump