Reconstitution of RND tripartite multidrug complexes for single-particle electron microscopy

Tripartite multidrug RND efflux pumps (TEP) made of an inner membrane RND transporter, an outer membrane factor (OMF) and a periplasmic adaptor protein (PAP) form a canal for expelling drugs through the cell wall of Gram-negative bacteria. Various RND transporters function with their cognate PAP and OMF although the rule is not so strict and an exchange with other OMF or PAP is not detrimental to TEP function. The molecular mechanism by which OMF-PAP-RND tripartite complexes are assembled is still poorly understood, as is the rule governing partner selection, leading to exchanges of TEP partners. Here, we present the reconstitution of the Pseudomonas aeruginosa OprM-MexA-MexB TEP and the chimeric OprN-MexA-MexB TEP into nanodiscs, combined with structural characterization by single-particle electron microscopy. This method allows assessment of the ability to form TEPs with cognate and non-cognate partners under controlled conditions, providing insight into the molecular recognition of the TEP components. The reconstitutions provide a better understanding of the effect of pH and other factors on TEP assembly.