Tripartite assembly of RND multidrug efflux pumps

Abstract

Tripartite multidrug efflux systems of Gram-negative bacteria are composed of an inner membrane transporter, an outer membrane channel and a periplasmic adaptor protein. They are assumed to form ducts inside the periplasm facilitating drug exit across the outer membrane. Here we present the reconstitution of native Pseudomonas aeruginosa MexAB–OprM and Escherichia coli AcrAB–TolC tripartite Resistance Nodulation and cell Division (RND) efflux systems in a lipid nanodisc system. Single-particle analysis by electron microscopy reveals the inner and outer membrane protein components linked together via the periplasmic adaptor protein. This intrinsic ability of the native components to self-assemble also leads to the formation of a stable interspecies AcrA–MexB–TolC complex suggesting a common mechanism of tripartite assembly. Projection structures of all three complexes emphasize the role of the periplasmic adaptor protein as part of the exit duct with no physical interaction between the inner and outer membrane components.

Nanodiscs in Membrane Biochemistry and Biophysics

“…211 Helices rearrange in the membrane with inter-helical loops forming a funnel for the entrance to the pore. Others have utilized Nanodiscs in electron microscopic investigations of lipoxygenase, 212 drug efflux pumps, 213 the magnesium channel, 214 AMP dependent protein kinase, 93 the ribosome-SecYE complex 215 and other membrane proteins. 119,216 Thus Nanodiscs and electron microscopy have become a mainstay for the structural determination of membrane proteins and their interactions to form supramolecular complexes involved in signaling, energy transduction and transport.…”

Efflux pump inhibitors of clinically relevant multidrug resistant bacteria

“…The available crystal structures for the individual subunits of the efflux pump have provided an insight into AcrAB‐TolC pumping mechanism . However, details of their interactions and stoichiometry, as well as how they are organized within the pump remained a matter of debate until the first pseudoatomic structure of tripartite efflux complex was published and the overall shape of the pump, as well as the relative arrangements of its components, revealed by electron microscopy studies . Just recently, the near‐atomic resolution cryoEM structures of the full AcrAB‐TolC assembly in both resting and drug transport states, with associated tertiary and quaternary structure changes, were reported in detail, providing a better understanding of the function of this efflux pump machinery .…”

“…Crystal structures have been reported for all three parts of the MexAB‐OprM efflux system, although the structure of an assembled tripartite complex has not yet been determined. However, the reconstitution of native MexAB‐OprM in a lipid nanodisc system has recently been reported, demonstrating the intrinsic ability of the native components to self‐assemble into a stable tripartite complex …”

“…Nevertheless, extensive work in this field in the last few decades has resulted in the generation of a lot of interesting information regarding the structure‐activity relationship (SAR) in different structural classes of EPIs targeting NorA and RND efflux pumps. Moreover, the known crystal structures of individual subunits of the tripartite efflux systems of E. coli AcrAB‐TolC and P. aeruginosa MexAB‐OprM, as well as the structures of their whole tripartite assemblies, offer an opportunity for the further exploration and design of novel EPIs according to available structural information …”

The antimicrobial activity of nanoparticles: present situation and prospects for the future

“…The main mechanisms with respect to biochemistry are 1) the alteration of targets; 2) the generation of inactivated enzymes or passivated enzymes; 3) the use of active efflux pump systems; 25,26 4) the presentation of obstacles to antibiotic permeation; 5) the formation of biofilms; 27 and 6) the emergence and elimination of a specific protein, such as BamA 28 or KatG, 29 which can affect infection through unknown mechanisms. Certain bacteria show antimicrobial resistance through only one of the mechanisms listed earlier, but two or more mechanisms can also be combined in one type of bacterium, including 7) induction of an antagonist through metabolic pathways or 8) increased production of a competitive inhibitor counteracting the antibiotic.…”