Crystal Structure of the Periplasmic Component of a Tripartite Macrolide-Specific Efflux Pump |
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Authors: | Soohwan Yum Yongbin Xu Shunfu Piao Hong-Man Kim Kyung-Jin Kim Min-Ho Jeong Kangseok Lee |
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Affiliation: | 1 College of Pharmacy and Research Institute for Drug Development, Pusan National University, Busan 609-735, Korea 2 Department of Life Science, Chung-Ang University, Seoul 156-756, Korea 3 Dong-A University Medical Science Research Center, Busan 602-714, Korea 4 Beamline Division, Pohang Accelerator Laboratory, Pohang 790-784, Korea 5 Division of Electron Microscopic Research, Korea Basic Science Institute, Daejeon 305-333, Korea 6 Biomedical Research Center, Korea Institute of Science and Technology, Seoul 136-791, Korea |
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Abstract: | In Gram-negative bacteria, type I protein secretion systems and tripartite drug efflux pumps have a periplasmic membrane fusion protein (MFP) as an essential component. MFPs bridge the outer membrane factor and an inner membrane transporter, although the oligomeric state of MFPs remains unclear. The most characterized MFP AcrA connects the outer membrane factor TolC and the resistance-nodulation-division-type efflux transporter AcrB, which is a major multidrug efflux pump in Escherichia coli. MacA is the periplasmic MFP in the MacAB-TolC pump, where MacB was characterized as a macrolide-specific ATP-binding-cassette-type efflux transporter. Here, we report the crystal structure of E. coli MacA and the experimentally phased map of Actinobacillus actinomycetemcomitans MacA, which reveal a domain orientation of MacA different from that of AcrA. Notably, a hexameric assembly of MacA was found in both crystals, exhibiting a funnel-like structure with a central channel and a conical mouth. The hexameric MacA assembly was further confirmed by electron microscopy and functional studies in vitro and in vivo. The hexameric structure of MacA provides insight into the oligomeric state in the functional complex of the drug efflux pump and type I secretion system. |
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Keywords: | MFP, membrane fusion protein RND, resistance-nodulation-division ABC, ATP-binding cassette Ec AcrA, Escherichia coli AcrA Aa MacA, Actinobacillus actinomycetemcomitans MacA MAD, multiple anomalous dispersion Ec MacA, Escherichia coli MacA MIC, minimal inhibitory concentration ORF, open reading frame |
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