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Mechanism of the stimulation of MacB ATPase by the periplasmic Membrane Fusion Protein (MFP) MacA
Author(s) -
Modali Sita D,
Zgurskaya Helen I
Publication year - 2010
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.24.1_supplement.699.5
Subject(s) - periplasmic space , bacterial outer membrane , chemistry , escherichia coli , biochemistry , efflux , atpase , proteolysis , inner membrane , mutant , microbiology and biotechnology , biology , membrane , enzyme , gene
Drug efflux pumps are the main contributors to the failure of antibiotic therapy. In Gram‐negative bacteria, these pumps function as tripartite complexes that extrude drugs out of the cells. In Escherichia coli, MacA‐MacB‐TolC is the first experimentally characterized macrolide specific efflux pump. Here, MacB is the ABC type inner membrane transporter, TolC is the outer membrane channel and MacA is the periplasmic Membrane Fusion Protein. MacA physically connects MacB to TolC and also stimulates the ATPase activity of MacB. The C‐terminal domain of MacA is essential for the MacB ATPase stimulatory activity. However, the biochemical mechanism of MacA mediated stimulation of MacB ATPase is unknown. In this study, using site directed mutagenesis we identified G353 and G357 residues in MacA which are essential for the stimulation of MacB ATPase in vitro. Trypsin proteolysis of the wild type MacA and mutants showed that mutations in these positions affect the conformation of MacA both in vivo and in vitro . Protein co‐purification and proteolytic digests indicate that MacA mutants assemble into the tripartite complex MacAB‐TolC. However, mutant complexes are different from those of the wild type MacA. We conclude that the amino acid residues G353 and G357 are essential for the function of MacA in stimulating the activity of MacB ATPase and also for the assembly of the proper MacA‐MacB‐TolC efflux pump. NIH Grant AI052293