Tripartite assembly of RND multidrug efflux pumps.

François Orange,Olivier Lambert,Laetitia Daury,Isabelle Broutin,Klaas M Pos,Jean-Christophe Taveau,Céline Gounou,Laura Monlezun,Alice Verchère,Martin Picard,Ravi K R Marreddy

doi:10.1038/ncomms10731

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.

Highlights

Tripartite multidrug efflux systems of Gram-negative bacteria are composed of an inner membrane transporter, an outer membrane channel and a periplasmic adaptor protein
The control of the assembly process relied on the insertion of a single molecule per ND, which necessitated the use of two membrane scaffold protein (MSP) differing in size (MSP1D1 or MSP1E3D1) because of the respective diameters of the transmembrane domains of the Resistance Nodulation and cell Division (RND) and outer membrane factor (OMF) proteins (RNDE80 Å, OMFE40–55 Å)
By means of a ND toolkit, integral membrane components of two tripartite RND efflux systems were incorporated into a lipid membrane ND and were further assembled after adding purified lipidated membrane fusion protein (MFP)

Summary

Introduction

Tripartite multidrug efflux systems of Gram-negative bacteria are composed of an inner membrane transporter, an outer membrane channel and a periplasmic adaptor protein. It has been postulated that drugs are transported from the periplasmic side across the outer membrane in an energy-dependent manner via the RND protein and the OMF channel[7,8,9,15,16,17,18,19,20,21] This intriguing transport mechanism is suggested to occur via a peristaltic mode through the protomers of the trimeric RND component caused by consecutive functional cycling of the protomers through three different states (loose, tight and open or access, binding and extrusion)[8,9,10,20,22,23,24]. The visualization by singleparticle EM reveals tripartite complexes made of the inner and outer membrane protein components linked together via the periplasmic adaptor protein emphasizing its role as part of the exit duct with no physical interaction between the inner and outer membrane components

Methods

Results

Conclusion