Functional Implications of an Intermeshing Cogwheel-like Interaction between TolC and MacA in the Action of Macrolide-specific Efflux Pump MacAB-TolC

Abstract

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 ␣-barrel to investigate the role of the TolC ␣-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 ␣-helical tip region. Electron microscopic study delineated that this complex was made by tip-to-tip interaction between the tip regions of the ␣-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.Drug resistance of microbial pathogens presents an increasing threat to public health (1). In Gram-negative pathogens, high levels of intrinsic or acquired drug resistance are conferred by three-component multidrug efflux pumps, which are composed of the inner membrane transporter, the outer membrane factor (OMF), and the periplasmic membrane fusion protein (MFP) 4 (2-5). These tripartite complexes span the entire twomembrane envelope of Gram-negative bacteria and expel various molecules into the medium, utilizing a proton gradient or ATP hydrolysis. The inner membrane transporters belong to one of three structurally dissimilar superfamilies of proteins: resistance-nodulation-cell division (RND), ATP-binding cassette (ABC), or major facilitator. The inner membrane transporters expel the substrates through the central channel of the OMF, as exemplified by Escherichia coli TolC, which spans the outer membrane (6). The MFP, which connects the other two components in the periplasm, is also essential for the function of the efflux pump.In E. coli, AcrAB-TolC acts as a major multidrug efflux pump (7-9), where AcrB is the RND-type inner membrane transporter and AcrA belongs to MFP. The homotrimeric TolC is embedded in the outer membrane and continues ϳ100 Å into the periplasmic space as an ␣-barrel composed of six ␣-hairpins that form the wall of a 35-Å inner-diameter cylindrical channel (10). The TolC channel is closed at the aperture end and the channel opening is induced only in the presence of the other components, the mechanism of which remains to be determined at the molecular level.The MacAB-TolC pump has been identified in E. coli; the inner membrane transporter MacB belongs to non-canonic ABC-type transporters (8,9,11,12), and MFP MacA shares structural similarity with AcrA (sequence similarity 44%) (13). Overproduction of MacAB results in increased resistance to the macrolide antibiotics in macrolide-susceptible AcrAB-deficient E. coli (8, 9, 11).The s...

Assembly and Channel Opening of Outer Membrane Protein in Tripartite Drug Efflux Pumps of Gram-negative Bacteria

“…MexA ␣-Barrel Domain Interacts with OprM ␣-Barrel Tip Region-To examine whether or not the MexA ␣-hairpin domain is capable of interacting with the ␣-barrel tip region of the cognate OEP OprM, as observed with AcrA and TolC, we constructed a chimeric protein containing the OprM ␣-barrel tip region based on the hexameric protein A. actinomycetemcomitans MacA, as used in construction of the TolC ␣-barrel tip region in the previous reports (14,31). The OprM monomer consists largely of two repeats, which contain one ␣-hairpin at the ␣-barrel tip region such as TolC, whereas A. actinomycetemcomitans MacA has only one ␣-hairpin per monomer.…”

“…However, it was not straightforward due to the integral membrane protein OprM, and the low affinity between the two proteins in the presence of the detergent that stabilizes the membrane protein. To overcome the problem, we had to construct the forced hexamer of MexA using the chimeric approach reported previously (14,31). This approach has been proven to be effective in representing the functional structure of ␣-hairpin domain of AcrA, a MexA homolog, in vivo and in vitro (14).…”

“…In the resulting three-dimensional electron density maps, this flexibility is represented by additional masses at one of the two ends. In previous reports, similar electron microscopic analyses were done with the TolC ␣-barrel tip and MacA (31), and the TolC ␣-barrel tip and AcrA ␣-hairpin (14).…”

“…The functional importance of the hexameric funnel structure also has been demonstrated (29,30). A chimeric protein containing the TolC ␣-barrel periplasmic end region formed a tight complex with MacA in an intermeshing cogwheel-to-cogwheel manner (30,31). Results from the chimeric studies suggested that the assembly mechanism of AcrA and TolC share that of MacA and TolC (13,14,31).…”

“…A chimeric protein containing the TolC ␣-barrel periplasmic end region formed a tight complex with MacA in an intermeshing cogwheel-to-cogwheel manner (30,31). Results from the chimeric studies suggested that the assembly mechanism of AcrA and TolC share that of MacA and TolC (13,14,31). Recently, the crystal structure of CusB in complex with CusA, which are the components of an RND-type tripartite pump CusB-CusACusC, revealed that the MFP CusB assumes a funnel-like hexameric assembly upon interaction with the RND-type transporter (19).…”