Pseudoatomic Structure of the Tripartite Multidrug Efflux Pump AcrAB-TolC Reveals the Intermeshing Cogwheel-like Interaction between AcrA and TolC

Jeong, Hyomin; Kim, Jin Sik; Song, Saemee; Shigematsu, Hideki; Yokoyama, Takeshi; Hyun, Jaekyung; Ha, Nam Chul

doi:10.1016/j.str.2015.12.007

Cited by 51 publications

(67 citation statements)

References 24 publications

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“…The increased diameter (from 13.5 to 29.0 Å) upon TolC-AcrA association, would admit the translocation of larger molecules, which is in agreement with other strong and recent experimental evidences. [17][18][19] The new proposed model also satisfied the Cryo-EM map volume described by Du et al 6 as seen in Figure 4c. Torres et al 2385 Vol.…”

Section: Resultssupporting

confidence: 69%

Alternative Model for RND-Type Efflux Pump

Tôrres¹,

Pascutti²,

Bisch³

et al. 2016

Journal of the Brazilian Chemical Society

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RND (resistance-nodulation-division) transporters are found in several Gram-negative bacteria species. These proteins form a complex along with membrane fusion proteins and outer membrane factors. This complex acts as an efflux pump, transporting several different molecules directly from the cytoplasm to the extracellular medium. Although the crystallographic structure of each protein of the complex is known, the complete complex assembly is not yet fully characterized. In 2014, a model for the complete system, based upon a cryoelectron-microscopy map, was proposed. In the present work, we propose an alternative model that also satisfies the volume obtained from the cryoelectron-microscopy assay. In this model, the AcrA helical domains are interleaved to the helical domains of the TolC protein, increasing the diameter of the formed pore. We believe that this model represents a better model for RND-type efflux pump and might contribute to the characterization of this system. Keywords: RND efflux system, comparative modeling, efflux pump, molecular modeling, protein structure IntroductionMulti-drug resistance systems, like the resistancenodulation-division RND-type efflux pump, play a crucial role on the adaptation of bacteria to toxic environments and are key components on cellular extrusion of structurally diverse antimicrobials in Gram-negative bacteria. 1These systems are composed of at least three different protein families. In Escherichia coli the AcrA-AcrB-TolC complex is a model system for the RND-type pump. AcrB is located at the inner membrane and is responsible for the translocation of several compounds from the cytoplasm to the periplasm, powered by a proton antiport mechanism. AcrA, located at the periplasm, acts as an adaptor protein, believed to assemble in a hexameric disposition. Finally, TolC forms a pore across the outer membrane, connecting the periplasm and the extracellular environment. Some authors have raised hypothesis regarding the assembly of the complex AcrAB-TolC, nevertheless the exact spatial conformation is still a matter of debate.3-5 In 2014, Du et al. 6 built AcrA-AcrB constructs and performed electron microscopy and cryo-electron microscopy assays that provided significant understanding of the complete pump assembly. It was proposed that TolC and AcrB are connected by the association with AcrA and that the former proteins make no direct contact, which is corroborated by cryo-electron microscopy density map. The model proposed by Du et al. 6 was in good agreement with previous works in what regarded the 3:6:3 stoichiometry ratio (AcrB:AcrA:TolC). 7 On the other hand, the proposed model had some very interesting novel features, as, for example, the fact that TolC and AcrB made no direct contact. The AcrA-TolC contact interface, as presented by the referred work, considered the TolC coiled-coils to be located inside the AcrA-formed pore lumen. This mode of interaction was based solely on the cryo-EM data, despite the fact that crystallographic data for the intermeshing cogwheel-t...

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Section: Resultssupporting

confidence: 69%

Alternative Model for RND-Type Efflux Pump

Tôrres¹,

Pascutti²,

Bisch³

et al. 2016

Journal of the Brazilian Chemical Society

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“…However, if the TriABC-OpmH complex resembles that of AcrABTolC, then in the assembled complex, the channel is in the open conformation, which is stabilized by specific interactions with MFPs ( Fig. 1C and 4C) (7,8). To investigate whether any of the disulfide bonds between MFPs and OpmH contribute to opening of the channel and prevent its closure, we measured the change in susceptibility to azithromycin, an ϳ740-Da antibiotic that does not readily permeate the outer membrane and is not a substrate of TriABC-OpmH.…”

Section: Figmentioning

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%

“…In this complex, the MFP AcrA assembles a hexameric, inverted-funnel-like structure that interacts with the periplasmic porter domain of the inner membrane transporter AcrB and the periplasmic tip of the OMF TolC (7,8). Amino acid residues at the hairpin loops of AcrA interact with the tip of the ␣-helical barrel of TolC, forcing its opening and stabilizing the open conformation.…”

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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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“…A small peptide, AcrZ, has been identified that modifies the activity of AcrB (Hobbs et al, 2012). Previous electron microscopy studies of the AcrAB-TolC pump have revealed the overall shape of the pump and the relative arrangement of its components (Daury et al, 2016; Du et al, 2014; Jeong et al, 2016). Due to the limited resolution, these studies were unable to identify the detailed interaction interfaces between the components.…”

Section: Introductionmentioning

confidence: 99%

An allosteric transport mechanism for the AcrAB-TolC multidrug efflux pump

Wang

Fan

Hryc

et al. 2017

eLife

207

358

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Bacterial efflux pumps confer multidrug resistance by transporting diverse antibiotics from the cell. In Gram-negative bacteria, some of these pumps form multi-protein assemblies that span the cell envelope. Here, we report the near-atomic resolution cryoEM structures of the Escherichia coli AcrAB-TolC multidrug efflux pump in resting and drug transport states, revealing a quaternary structural switch that allosterically couples and synchronizes initial ligand binding with channel opening. Within the transport-activated state, the channel remains open even though the pump cycles through three distinct conformations. Collectively, our data provide a dynamic mechanism for the assembly and operation of the AcrAB-TolC pump.DOI: http://dx.doi.org/10.7554/eLife.24905.001

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Pseudoatomic Structure of the Tripartite Multidrug Efflux Pump AcrAB-TolC Reveals the Intermeshing Cogwheel-like Interaction between AcrA and TolC

Cited by 51 publications

References 24 publications

Alternative Model for RND-Type Efflux Pump

Alternative Model for RND-Type Efflux Pump

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

An allosteric transport mechanism for the AcrAB-TolC multidrug efflux pump

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