In Gram-negative pathogens, multidrug efflux pumps that provide clinically significant levels of antibiotic resistance function as threecomponent complexes. They are composed of the inner membrane transporters belonging to one of three superfamilies of proteins, RND, ABC, or MF; periplasmic proteins belonging to the membrane fusion protein (MFP) family; and outer membrane channels exemplified by the Escherichia coli TolC. The three-component complexes span the entire two-membrane envelope of Gram-negative bacteria and expel toxic molecules from the cytoplasmic membrane to the medium. The architecture of these complexes is expected to vary significantly because of the structural diversity of the inner membrane transporters. How the three-component pumps are assembled, their architecture, and their dynamics remain unclear. In this study, we reconstituted interactions and compared binding kinetics of the E. coli TolC with AcrA, MacA, and EmrA, the periplasmic MFPs that function in multidrug efflux with transporters from the RND, ABC, and MF superfamilies, respectively. By using surface plasmon resonance, we demonstrate that TolC interactions with MFPs are highly dynamic and sensitive to pH. The affinity of TolC to MFPs decreases in the order MacA > EmrA > AcrA. We further show that MFPs are prone to oligomerization, but differ dramatically from each other in oligomerization kinetics and stability of oligomers. The propensity of MFPs to oligomerize correlates with the stability of MFP-TolC complexes and structural features of inner membrane transporters. We propose that recruitment of TolC by various MFPs is determined not only by kinetics of MFP-TolC interactions but also by oligomerization kinetics of MFPs and pH. multidrug efflux transporters ͉ protein-protein interactions ͉ surface plasmon resonance ͉ antibiotic resistance ͉ gram-negative envelope

In gram-negative bacteria, transporters belonging to the resistance-nodulation-cell division (RND) superfamily of proteins are responsible for intrinsic multidrug resistance. Haemophilus influenzae, a gram-negative pathogen causing respiratory diseases in humans and animals, constitutively produces the multidrug efflux transporter AcrB (AcrB HI ). Similar to other RND transporters AcrB HI associates with AcrA HI , the periplasmic membrane fusion protein, and the outer membrane channel TolC HI . Here, we report that AcrAB HI confers multidrug resistance when expressed in Escherichia coli and requires for its activity the E. coli TolC (TolC EC ) protein. To investigate the intracellular dynamics of AcrAB HI , single cysteine mutations were constructed in AcrB HI in positions previously identified as important for substrate recognition. The accessibility of these strategically positioned cysteines to the hydrophilic thiol-reactive fluorophore fluorescein-5-maleimide (FM) was studied in vivo in the presence of various substrates of AcrAB HI and in the presence or absence of AcrA HI and TolC EC . We report that the reactivity of specific cysteines with FM is affected by the presence of some but not all substrates. Our results suggest that substrates induce conformational changes in AcrB HI .Multidrug efflux transporters belonging to the resistancenodulation-cell division (RND) superfamily of proteins are broadly represented in gram-negative bacteria. Previous studies established that these transporters play a major role in the intrinsic and induced resistance of gram-negative bacteria to multiple antimicrobial agents including clinically important antibiotics (reviewed in references 7, 11, 18, and 27). Their contribution to antibiotic resistance in clinical settings made RND-type transporters attractive targets of a search for specific and broad-spectrum inhibitors that could be used to increase the efficacy of antibiotics (8).RND-type multidrug transporters from various species share a high degree of sequence and, correspondingly, structure conservation. Structural and functional studies showed that RNDtype transporters exist as trimers, which are organized into two large domains: the transmembrane hydrophobic domain comprised by 36 transmembrane ␣-helices (TMS) with each protomer contributing 12 TMSs and the large periplasmic domain exposed to aqueous environment of the periplasm (17). This impressive structure, however, is not sufficient to provide multidrug resistance. RND transporters associate with two accessory proteins. The periplasmic membrane fusion proteins (MFPs) are believed to mediate the interaction between an RND transporter and an outer membrane channel belonging to the outer membrane factor family of proteins (18,27). Together, these interacting three components span the inner and outer membranes and the periplasm of gram-negative bacteria. Mutations in any of the three proteins completely abolish intrinsic multidrug resistance of gram-negative bacteria.In the three-component complexes, RND transpor...