Mechanism of coupling drug transport reactions located in two different membranes

Zgurskaya, Helen I.; Weeks, Jon W.; Ntreh, Abigail T.; Nickels, Logan M.; Wolloscheck, David

doi:10.3389/fmicb.2015.00100

Cited by 53 publications

(56 citation statements)

References 99 publications

(178 reference statements)

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“…They claimed that the dimer is the entity that binds to TolC and that AcrA functions as a dimer (41). Those reports supported a 1:2:1 stoichiometry.…”

Section: Discussionsupporting

confidence: 59%

“…These experiments indicated that the ␣-hairpins of AcrA interact with TolC and that the other three domains of AcrA interact with the DN and PN2 subdomains of AcrB. On the other hand, surface plasmon resonance studies indicated that AcrA exists as a dimer (39,40) and that the dimer is the entity that binds to TolC, suggesting 1:2:1 stoichiometry in vitro (41). Recently, 3:6:3 models were also proposed on the basis of electron microscopy (EM) images of the AcrAB-TolC complex.…”

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confidence: 99%

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AcrB-AcrA Fusion Proteins That Act as Multidrug Efflux Transporters

et al. 2016

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The AcrAB-TolC system in Escherichia coli is an intrinsic RND-type multidrug efflux transporter that functions as a tripartite complex of the inner membrane transporter AcrB, the outer membrane channel TolC, and the adaptor protein AcrA. Although the crystal structures of each component of this system have been elucidated, the crystal structure of the whole complex has not been solved. The available crystal structures have shown that AcrB and TolC function as trimers, but the number of AcrA molecules in the complex is now under debate. Disulfide chemical cross-linking experiments have indicated that the stoichiometry of AcrB-AcrA-TolC is 1:1:1; on the other hand, recent cryo-electron microscopy images of AcrAB-TolC suggested a 1:2:1 stoichiometry. In this study, we constructed 1:1-fixed AcrB-AcrA fusion proteins using various linkers. Surprisingly, all the 1:1-fixed linker proteins showed drug export activity under both acrAB-deficient conditions and acrAB acrEF double-pump-knockout conditions regardless of the lengths of the linkers. Finally, we optimized a shorter linker lacking the conformational freedom imparted by the AcrB C terminus. These results suggest that a complex with equal amounts of AcrA and AcrB is sufficient for drug export function. IMPORTANCEThe structure and stoichiometry of the RND-type multidrug exporter AcrB-AcrA-TolC complex are still under debate. Recently, electron microscopic images of the AcrB-AcrA-TolC complex have been reported, suggesting a 1:2:1 stoichiometry. However, we report here that the AcrB-AcrA 1:1 fusion protein is active for drug export under acrAB-deficient conditions and also under acrAB acrEF double-deficient conditions, which eliminate the aid of free AcrA and its close homolog AcrE, indicating that the AcrB-AcrA 1:1 stoichiometry is enough for drug export function. In addition, the AcrB-AcrA fusion protein can function without the aid of free AcrA. We believe that these results are very important for considering the structure and mechanism of AcrABTolC-mediated multidrug export.

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“…They claimed that the dimer is the entity that binds to TolC and that AcrA functions as a dimer (41). Those reports supported a 1:2:1 stoichiometry.…”

Section: Discussionsupporting

confidence: 59%

mentioning

confidence: 99%

AcrB-AcrA Fusion Proteins That Act as Multidrug Efflux Transporters

et al. 2016

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“…The lipoyl, ␤-barrel, and membrane-proximal domains are made primarily of ␤-strands. The latter domains stabilize interactions with the transporter protein (6,9).…”

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confidence: 99%

“…In the resting state, the aperture of the OMF remains in a closed state and only upon functional interactions with the inner membrane components of the tripartite complex does the OMF transition into an open state, allowing efflux of substrates into the extracellular milieu (4)(5)(6)(7)(8) (Fig. 1C).…”

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confidence: 99%

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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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Electrophysiology of Bacterial Translocons

Pham

Delcour

2015

Springer Series in Biophysics

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Mechanism of coupling drug transport reactions located in two different membranes

Cited by 53 publications

References 99 publications

AcrB-AcrA Fusion Proteins That Act as Multidrug Efflux Transporters

AcrB-AcrA Fusion Proteins That Act as Multidrug Efflux Transporters

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

Electrophysiology of Bacterial Translocons

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