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Role of ATP binding and hydrolysis in assembly of MacAB – TolC macrolide transporter
Author(s) -
Lu Shuo,
Zgurskaya Helen I.
Publication year - 2012
Publication title -
molecular microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.857
H-Index - 247
eISSN - 1365-2958
pISSN - 0950-382X
DOI - 10.1111/mmi.12046
Subject(s) - periplasmic space , atp hydrolysis , bacterial outer membrane , biology , biochemistry , atpase , transporter , membrane protein , microbiology and biotechnology , escherichia coli , membrane , enzyme , gene
Summary MacB is a founding member of the M acrolide E xporter family of transporters belonging to the ATP ‐ B inding C assette superfamily. These proteins are broadly represented in genomes of both G ram‐positive and G ram‐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 G ram‐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.