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.
Highlights
Since the discovery of the first antibiotic drug penicillin in 1928, many antibiotics have been found and manufactured to treat infectious diseases (Nikaido 2009)
Since MacB variants are in different ATP hydrolysis state, we investigated whether MacBWT, MacB169, MacBYC and MacBLPL are functional in transport of macrolides
One of the reasons why Gram-negative bacteria are more resistant to antibiotics than Gram-positive bacteria is the distinctive structural feature of their cell wall
Summary
Since the discovery of the first antibiotic drug penicillin in 1928, many antibiotics have been found and manufactured to treat infectious diseases (Nikaido 2009). In Gram-negative bacteria, the presence of outer membrane bilayer and the expression of MDR efflux systems contribute to the intrinsic drug resistance (Lubelski, Konings et al 2007). Structural comparison between the PCD of MacB and the periplasmic domain of the RND transporter AcrB reveals some similarities The overall structure of TolC is a tapered hollow cylinder with a length of 140 Å It comprises a 40 Å long outer membrane β-barrel domain (the channel domain) and a contiguous 100 Å long α-helical barrel (the tunnel domain) projecting across the periplasmic space. Unlike other outer membrane proteins which contain one β-barrel per monomer, the channel domain of TolC exists as a distinct structure with 12 right-twisted β-strands forming a single β-barrel (Pautsch and Schulz 1998). The weakening of the intramonomer and intermonomer links relaxes the inner coiled-coils from their constrained position to form the open conformation (Koronakis, Sharff et al 2000; Andersen, Koronakis et al 2002) (Figure I.7 C)
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