Molecular Determinants for OMF Selectivity in Tripartite RND Multidrug Efflux Systems

Tripartite multidrug RND efflux systems made of an inner membrane transporter, an outer membrane factor (OMF) and a periplasmic adaptor protein (PAP) form a canal to expel drugs across Gram-negative cell wall. Structures of MexA-MexB-OprM and AcrA-AcrB-TolC, from Pseudomonas aeruginosa and Escherichia coli, respectively, depict a reduced interfacial contact between OMF and PAP, making unclear the comprehension of how OMF is recruited. Here, we show that a Q93R mutation of MexA located in the α-hairpin domain increases antibiotic resistance in the MexA_Q93R-MexB-OprM-expressed strain. Electron microscopy single-particle analysis reveals that this mutation promotes the formation of tripartite complexes with OprM and non-cognate components OprN and TolC. Evidence indicates that MexA_Q93R self-assembles into a hexameric form, likely due to interprotomer interactions between paired R93 and D113 amino acids. C-terminal deletion of OprM prevents the formation of tripartite complexes when mixed with MexA and MexB components but not when replacing MexA with MexA_Q93R. This study reveals the Q93R MexA mutation and the OprM C-terminal peptide as molecular determinants modulating the assembly process efficacy with cognate and non-cognate OMFs, even though they are outside the interfacial contact. It provides insights into how OMF selectivity operates during the formation of the tripartite complex.