Macrolide-specific efflux pump MacAB-TolC has been identified in diverse gram-negative bacteria including Escherichia coli. The inner membrane transporter MacB requires the outer membrane factor TolC and the periplasmic adaptor protein MacA to form a functional tripartite complex. In this study, we used a chimeric protein containing the tip region of the TolC α-barrel to investigate the role of the TolC α-barrel tip region with regard to its interaction with MacA. The chimeric protein formed a stable complex with MacA, and the complex formation was abolished by substitution at the functionally essential residues located at the MacA α-helical tip region. Electron microscopic study delineated that this complex was made by tip-to-tip interaction between the tip regions of the α-barrels of TolC and MacA, which correlated well with the TolC and MacA complex calculated by molecular dynamics. Taken together, our results demonstrate that the MacA hexamer interacts with TolC in a tip-to-tip manner, and implies the manner by which MacA induces opening of the TolC channel.
"olC using a chimeric protein from A . actinomycetemco - mmi - tans ( Aa ) , MacA , that contains TolC - barrel tip region , which we referred to as MacA - TolC - hybrid - dimer ( Xu et al . , 2011a ; 2011b ; 2012 ) . Because the MacA - TolC - hybrid - dimer protein exhibited a strong affinity to MacA and AcrA in vitro [ Kd value of ~0 . 7 nM ( Xu et al . , 2011b ) ] , this hybrid protein was considered to exhibit a TolC structure that specifically binds AcrA or MacA ."
[Show abstract][Hide abstract] ABSTRACT: Escherichia coli AcrAB-TolC is a multidrug efflux pump that expels a wide range of toxic substrates. The dynamic nature of the binding or low affinity between the components has impeded elucidation of how the three components assemble in the functional state. Here, we created fusion proteins composed of AcrB, a transmembrane linker, and two copies of AcrA. The fusion protein exhibited acridine pumping activity, suggesting that the protein reflects the functional structure in vivo. To discern the assembling mode with TolC, the AcrBA fusion protein was incubated with TolC or a chimeric protein containing the TolC aperture tip region. Three-dimensional structures of the complex proteins were determined through transmission electron microscopy. The overall structure exemplifies the adaptor bridging model, wherein the funnel-like AcrA hexamer forms an intermeshing cogwheel interaction with the α-barrel tip region of TolC, and a direct interaction between AcrB and TolC is not allowed. These observations provide a structural blueprint for understanding multidrug resistance in pathogenic Gram-negative bacteria.
Molecules and Cells 02/2015; 38(2):180-6. DOI:10.14348/molcells.2015.2277 · 2.09 Impact Factor
"As shown in Fig. 1C, the TolCV1 and TolCV2 proteins were cross-linked to MacA, whereas TolCV1 and TolCV2 variants were not, indicating that the TolCV1 and TolCV2 aperture region makes specific contact with MacA. Co-expressed TolC and MacA resulted in strong copurification as reported previously (Xu et al., 2011b). These results show that the TolCV1 and TolCV2 aperture tip regions are not only functionally related, but physically interact with MacA. "
[Show abstract][Hide abstract] ABSTRACT: TolC and its homologous family of proteins are outer membrane factors that are essential for exporting small molecules and toxins across the outer membrane in Gram-negative bacteria. Two open reading frames in the Vibrio vulnificus genome that encode proteins homologous to Escherichia coli TolC, designated TolCV1 and TolCV2, have 51.3% and 29.6% amino acid identity to TolC, respectively. In this study, we show that TolCV1 and TolCV2 functionally and physically interacted with the membrane fusion protein, MacA, a component of the macrolide-specific MacAB-TolC pump of E. coli. We further show that the conserved residues located at the aperture tip region of the α-hairpin of TolCV1 and TolCV2 played an essential role in the formation of the functional MacAB-TolC pump using site-directed mutational analyses. Our findings suggest that these outer membrane factors have conserved tip-to-tip interaction with the MacA membrane fusion protein for action of the drug efflux pump in Gramnegative bacteria.
The Journal of Microbiology 04/2013; 51(2):154-9. DOI:10.1007/s12275-013-2699-3 · 1.44 Impact Factor
"The affinity of PBPs to transporters changes with the availability of substrate and conformational transitions in both PBPs and transporters are linked to hydrolysis of ATP. In contrast, MacA exists as a hexamer, which presumably forms a tunnel-like structure covering the periplasmic exit of MacB (Yum et al., 2009; Xu et al., 2011). The cytoplasmic region of MacA is limited to 10 amino acid residues, seven of which are either lysine or arginine. "
[Show abstract][Hide abstract] ABSTRACT: MacB is a founding member of the Macrolide Exporter family of transporters belonging to the ATP-Binding Cassette superfamily. These proteins are broadly represented in genomes of both Gram-positive and Gram-negative bacteria and are implicated in virulence and protection against antibiotics and peptide toxins. MacB transporter functions together with MacA, a periplasmic membrane fusion protein, which stimulates MacB ATPase. In Gram-negative bacteria, MacA is believed to couple ATP hydrolysis to transport of substrates across the outer membrane through a TolC-like channel. In this study, we report a real-time analysis of concurrent ATP hydrolysis and assembly of MacAB-TolC complex. MacB binds nucleotides with a low millimolar affinity and fast on- and off-rates. In contrast, MacA-MacB complex is formed with a nanomolar affinity, which further increases in the presence of ATP. Our results strongly suggest that association between MacA and MacB is stimulated by ATP binding to MacB but remains unchanged during ATP hydrolysis cycle. We also found that the large periplasmic loop of MacB plays the major role in coupling reactions separated in two different membranes. This loop is required for MacA-dependent stimulation of MacB ATPase and at the same time, contributes to recruitment of TolC into a trans-envelope complex.
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