The tip region of the MacA α-hairpin is important for the binding to TolC to the Escherichia coli MacAB–TolC pump

Xu, Yongbin; Sim, Se-Hoon; Song, Saemee; Piao, Shunfu; Kim, Hong-Man; Jin, Xiao Ling; Lee, Kangseok; Ha, Nam‐Chul

doi:10.1016/j.bbrc.2010.03.097

Cited by 40 publications

(78 citation statements)

References 24 publications

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“…We also found that TriA containing a single point mutation in the ␣-helical hairpin domain, TriA R130D , but not its counterpart TriB R118D , lost its functional interaction with E. coli TolC. The analogous residues in homohexameric MFPs, such as AcrA or MacA, are also functionally important and are thought to form an interface with TolC (18,19). To determine whether the same interactions are important for the engagement and function with OpmH, pBSPII derivatives expressing wild-type TriABC or TriAxBC and the mutant variant TriA R130D or TriB R118D were transformed into JWW9 (attTn7::P BAD -opmH His ⌬ompH::Gm) cells and drug susceptibility and copurification assays were carried out to analyze the pump's functionality and its assembly.…”

Section: Resultsmentioning

confidence: 77%

Opening the Channel: the Two Functional Interfaces of Pseudomonas aeruginosa OpmH with the Triclosan Efflux Pump TriABC

et al. 2016

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TriABC-OpmH is an efflux pump from Pseudomonas aeruginosa with an unusual substrate specificity and protein composition. When overexpressed, this pump confers a high level of resistance to the biocide triclosan and the detergent SDS, which are commonly used in combinations for antimicrobial treatments. This activity requires an RND transporter (TriC), an outer membrane channel (OpmH), and two periplasmic membrane fusion proteins (TriA and TriB) with nonequivalent functions. In the active complex, TriA is responsible for the recruitment of OpmH, while TriB is responsible for stimulation of the transporter TriC. Here, we used the functional and structural differences between the two membrane fusion proteins to link their functional roles to specific interactions with OpmH. Our results provide evidence that the TriB-dependent stimulation of the TriC transporter is coupled to opening of the OpmH aperture through binding to the interprotomer groove of OpmH. IMPORTANCEMultidrug efflux transporters are important contributors to intrinsic and acquired antibiotic resistance in clinics. In Gram-negative bacteria, these transporters have a characteristic tripartite architecture spanning the entire two-membrane cell envelope. How such complexes are assembled and how the reactions separated in two different membranes are coupled to provide efficient efflux of various compounds across the cell envelope remain unclear. This study addressed these questions, and the results suggest a mechanism for functional integration of drug efflux by the inner membrane transporter and opening of the channel for transport across the outer membrane. In Gram-negative bacteria, polyspecific transporters detoxify the inner membrane and periplasm of noxious compounds and contribute to clinical antibiotic resistance (1). A characteristic structural feature of these transporters is the formation of tripartite complexes spanning both the inner and outer membranes of Gram-negative cell envelopes. Located in the outer membrane are proteins belonging to the outer membrane factor (OMF) family that act as channels for substrate expulsion across the low-permeability barrier of the outer membrane. OMFs show very little sequence similarity to one another but are structurally similar (2). OMF structures comprise both a ␤-barrel domain, a common structural feature of other outer membrane proteins, and a large ␣-helical barrel (3) (Fig. 1A). The periplasmic tip of the ␣-helical barrel converges to form a closed aperture that prevents leakage of the periplasmic content and uptake of various molecules from the external medium (Fig. 1B). Large extracellular loops on the external side of the ␤-barrel domain also help with blocking noxious chemicals from entering the cell. At the aperture, a series of ionic bridges act as the locking gate of the aperture and disruption of these causes a "leaky" phenotype (4, 5). A secondary gate of aspartate or, in a few cases, a ring of hydrophobic residues acts additionally to keep the aperture locked.In the resting state,...

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Section: Resultsmentioning

confidence: 77%

Opening the Channel: the Two Functional Interfaces of Pseudomonas aeruginosa OpmH with the Triclosan Efflux Pump TriABC

et al. 2016

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“…1A). Because only ␣-hairpin domains of MFPs are involved in the binding to the cognate OEPs (17,30), the MexA ␣-hairpin domain in the chimeric protein should be suitable for investigating the interaction with OprM. n Improved Binding Model for MexA and OprM…”

Section: Resultsmentioning

confidence: 99%

“…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).…”

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confidence: 95%

“…The crystal structure of the MFP MacA revealed a funnel-like hexameric assembly, where the six ␣-hairpin domains from the hexamer pack to form a hollow cylinder (29). 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).…”

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confidence: 98%

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

Moeller

Jun

et al. 2012

Journal of Biological Chemistry

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Background: Pseudomonas aeruginosa mainly achieves multidrug resistance by use of the MexAB-OprM pump. Results: We determined the crystal structure of MexA. Electron microscopy work using MexA and OprM reveals that MexA makes a tip-to-tip interaction with OprM. Conclusion:We suggest an assembly and channel opening model for the pump. Significance: This study provides a better understanding of multidrug resistance in Gram-negative bacteria.

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“…A "bridging model" was proposed for the TolC-DevBCA homologue TolC-MacAB, with a MacA hexamer fitting to the tip of TolC in a cogwheel-like manner (9,40,41). It was derived from in silico protein models based on MacA crystals resolved to hexamers and electron micrographs showing a barrel-like hexameric assembly.…”

Section: Discussionmentioning

confidence: 99%

Novel ATP-driven Pathway of Glycolipid Export Involving TolC Protein

Staron

Forchhammer

Maldener

2011

Journal of Biological Chemistry

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Background:The ABC exporter DevBCA and the outer membrane protein TolC are necessary for maturation of heterocysts in filamentous cyanobacteria. Results: DevBCA-TolC form an ATP-driven efflux pump and export heterocyst-specific glycolipids. Conclusion: DevBCA-TolC provide a novel pathway for glycolipid export. Significance: Mechanistic details of efflux pumps/ABC exporters are important for understanding various bacterial processes such as cell differentiation, acclimatization processes, or drug resistance.

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The tip region of the MacA α-hairpin is important for the binding to TolC to the Escherichia coli MacAB–TolC pump

Cited by 40 publications

References 24 publications

Opening the Channel: the Two Functional Interfaces of Pseudomonas aeruginosa OpmH with the Triclosan Efflux Pump TriABC

Opening the Channel: the Two Functional Interfaces of Pseudomonas aeruginosa OpmH with the Triclosan Efflux Pump TriABC

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

Novel ATP-driven Pathway of Glycolipid Export Involving TolC Protein

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