Structures and transport dynamics of a Campylobacter jejuni multidrug efflux pump

Kanagalaghatta R Rajashankar,Nitin Kumar,Chih-Chia Su,Jared A Delmar,Linxiang Yin,Yeon-Kyun Shin,Jani Reddy Bolla,Hsiang-Ting Lei,Qijing Zhang,Lei Dai,Tsung-Han Chou,Edward W Yu,Abhijith Radhakrishnan

doi:10.1038/s41467-017-00217-z

Abstract

Resistance-nodulation-cell division efflux pumps are integral membrane proteins that catalyze the export of substrates across cell membranes. Within the hydrophobe-amphiphile efflux subfamily, these resistance-nodulation-cell division proteins largely form trimeric efflux pumps. The drug efflux process has been proposed to entail a synchronized motion between subunits of the trimer to advance the transport cycle, leading to the extrusion of drug molecules. Here we use X-ray crystallography and single-molecule fluorescence resonance energy transfer imaging to elucidate the structures and functional dynamics of the Campylobacter jejuni CmeB multidrug efflux pump. We find that the CmeB trimer displays a very unique conformation. A direct observation of transport dynamics in individual CmeB trimers embedded in membrane vesicles indicates that each CmeB subunit undergoes conformational transitions uncoordinated and independent of each other. On the basis of our findings and analyses, we propose a model for transport mechanism where CmeB protomers function independently within the trimer.

Highlights

Resistance-nodulation-cell division efflux pumps are integral membrane proteins that catalyze the export of substrates across cell membranes
This tripartite system is composed of the CmeB efflux pump, an inner membrane resistance-nodulation-cell division (RND)[12] transport protein that contains substrate-binding sites and transduces the electrochemical energy required for pumping drugs out of the cell; the CmeA periplasmic protein, a member of the membrane fusion protein family; and the CmeC outer membrane-associated protein that is integral to the outer membrane
To understand the transport mechanism of the CmeB efflux pump from C. jejuni, we here define the X-ray structures of this membrane protein, which assembles as a trimer

Summary

Introduction

Resistance-nodulation-cell division efflux pumps are integral membrane proteins that catalyze the export of substrates across cell membranes. The Cme locus consists of three tandemly linked genes (cmeABC) encoding protein components of the tripartite Cme efflux pump (CmeA, CmeB, and CmeC), where all three components are absolutely required for substrate expulsion This tripartite system is composed of the CmeB efflux pump, an inner membrane resistance-nodulation-cell division (RND)[12] transport protein that contains substrate-binding sites and transduces the electrochemical energy required for pumping drugs out of the cell; the CmeA periplasmic protein, a member of the membrane fusion protein family; and the CmeC outer membrane-associated protein that is integral to the outer membrane. Using singlemolecule fluorescence resonance energy transfer (sm-FRET) imaging, we demonstrate that each CmeB protomer within the trimer is able to function independently

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Conclusion