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Structural basis of RND-type multidrug exporters.

Yamaguchi A, Nakashima R, Sakurai K - Front Microbiol (2015)

Bottom Line: Multidrug recognition is based on a multisite drug-binding mechanism, in which two voluminous multidrug-binding pockets in cell membrane exporters recognize a wide range of substrates as a result of permutations at numerous binding sites that are specific for the partial structures of substrate molecules.Substrates are transported through dual multidrug-binding pockets via the peristaltic motion of the substrate translocation channel.Although there are no clinically available inhibitors of bacterial multidrug exporters, efforts to develop inhibitors based on structural information are underway.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Cell Membrane Structural Biology, Institute of Scientific and Industrial Research, Osaka University Ibaraki, Japan.

ABSTRACT
Bacterial multidrug exporters are intrinsic membrane transporters that act as cellular self-defense mechanism. The most notable characteristics of multidrug exporters is that they export a wide range of drugs and toxic compounds. The overexpression of these exporters causes multidrug resistance. Multidrug-resistant pathogens have become a serious problem in modern chemotherapy. Over the past decade, investigations into the structure of bacterial multidrug exporters have revealed the multidrug recognition and export mechanisms. In this review, we primarily discuss RND-type multidrug exporters particularly AcrAB-TolC, major drug exporter in Gram-negative bacteria. RND-type drug exporters are tripartite complexes comprising a cell membrane transporter, an outer membrane channel and an adaptor protein. Cell membrane transporters and outer membrane channels are homo-trimers; however, there is no consensus on the number of adaptor proteins in these tripartite complexes. The three monomers of a cell membrane transporter have varying conformations (access, binding, and extrusion) during transport. Drugs are exported following an ordered conformational change in these three monomers, through a functional rotation mechanism coupled with the proton relay cycle in ion pairs, which is driven by proton translocation. Multidrug recognition is based on a multisite drug-binding mechanism, in which two voluminous multidrug-binding pockets in cell membrane exporters recognize a wide range of substrates as a result of permutations at numerous binding sites that are specific for the partial structures of substrate molecules. The voluminous multidrug-binding pocket may have numerous binding sites even for a single substrate, suggesting that substrates may move between binding sites during transport, an idea named as multisite-drug-oscillation hypothesis. This hypothesis is consistent with the apparently broad substrate specificity of cell membrane exporters and their highly efficient ejection of drugs from the cell. Substrates are transported through dual multidrug-binding pockets via the peristaltic motion of the substrate translocation channel. Although there are no clinically available inhibitors of bacterial multidrug exporters, efforts to develop inhibitors based on structural information are underway.

No MeSH data available.


Related in: MedlinePlus

Intramolecular water-accessible channels in the AcrB trimer (Nakashima et al., 2011). The channels are shown as colored solid surfaces, as calculated using the CAVER program (Medek et al., 2007). The proximal pocket, distal pocket, entrances and funnel-like exit are depicted in green, pink, gray, and yellow, respectively. The channel apertures at the entrance and exit are depicted in black. (1) Inner membrane entrance (Murakami et al., 2006), (2) periplasmic entrance (Seeger et al., 2006), (3) central cavity entrance (Nakashima et al., 2011). (A) Side view. The channels in the access monomer behind the figure have been omitted. The central cavity and central hole are depicted as dotted lines. (B) Horizontal cut view of the porter domain. The yellow circle indicates the closed pore-like structure comprising three central α-helices (depicted as a ribbon model with dense color), which was postulated to be a part of the putative substrate translocation channel during the early stages, however, it was not the case. The central α-helix of extrusion monomer is inclined 15° toward binding monomer more than the other two α-helices and, as a result, blocks the exit from the drug binding site. Bound minocycline (cyan), doxorubicin (orange), rifampicin (magenta), and erythromycin (yellow) overlap in the space-filling model.
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Figure 5: Intramolecular water-accessible channels in the AcrB trimer (Nakashima et al., 2011). The channels are shown as colored solid surfaces, as calculated using the CAVER program (Medek et al., 2007). The proximal pocket, distal pocket, entrances and funnel-like exit are depicted in green, pink, gray, and yellow, respectively. The channel apertures at the entrance and exit are depicted in black. (1) Inner membrane entrance (Murakami et al., 2006), (2) periplasmic entrance (Seeger et al., 2006), (3) central cavity entrance (Nakashima et al., 2011). (A) Side view. The channels in the access monomer behind the figure have been omitted. The central cavity and central hole are depicted as dotted lines. (B) Horizontal cut view of the porter domain. The yellow circle indicates the closed pore-like structure comprising three central α-helices (depicted as a ribbon model with dense color), which was postulated to be a part of the putative substrate translocation channel during the early stages, however, it was not the case. The central α-helix of extrusion monomer is inclined 15° toward binding monomer more than the other two α-helices and, as a result, blocks the exit from the drug binding site. Bound minocycline (cyan), doxorubicin (orange), rifampicin (magenta), and erythromycin (yellow) overlap in the space-filling model.

Mentions: Three monomers of AcrB are tightly interacted to form a jelly-fish or mushroom-like structure (Figure 4A). Long hairpins are deeply inserted into the next monomers to form the shape that is likened to the figure called “it takes three to tango” (Lomovslaya et al., 2002). Three PN1 subdomains form a core for the headpiece and the three central α-helices form a pore-like structure at the center of the trimer (Figure 4B). The funnel-like 30 Å opening at the top of the TolC-docking domain is the same size as the bottom of the TolC channel (Koronakis et al., 2000). In the transmembrane domain, there is a central hole of 30 Å diameter surrounded by three 12-α-helix bundles (Figure 4C). The central hole is not a water-filled channel: it is filled with a phospholipid bilayer (Nakashima et al., 2013). There is a central cavity on the putative surface of the phospholipid bilayer in the central hole and below the closed pore-like structure (Figure 5A). The central cavity comprises three windows, named vestibules, to the surface of the inner membrane (Figures 5, 6). Initially, this central cavity was identified as a substrate binding site, at which the substrates are taken up through vestibules from the outer leaflet of the phospholipid bilayer membrane, bound to the central cavity, transferred through the central pore when it is open, and ultimately extruded from the funnel-like exit at the top of the AcrB trimer into the TolC channel (Murakami et al., 2002). Although the substrate-binding structures of the three-fold symmetry crystals in the central cavity were reported in 2003 (Yu et al., 2003), subsequent studies have not been able to identify significant electron densities of bound drugs in the central cavity [Pos et al. (2004); Murakami et al. (unpublished observation)] of the symmetric crystal.


Structural basis of RND-type multidrug exporters.

Yamaguchi A, Nakashima R, Sakurai K - Front Microbiol (2015)

Intramolecular water-accessible channels in the AcrB trimer (Nakashima et al., 2011). The channels are shown as colored solid surfaces, as calculated using the CAVER program (Medek et al., 2007). The proximal pocket, distal pocket, entrances and funnel-like exit are depicted in green, pink, gray, and yellow, respectively. The channel apertures at the entrance and exit are depicted in black. (1) Inner membrane entrance (Murakami et al., 2006), (2) periplasmic entrance (Seeger et al., 2006), (3) central cavity entrance (Nakashima et al., 2011). (A) Side view. The channels in the access monomer behind the figure have been omitted. The central cavity and central hole are depicted as dotted lines. (B) Horizontal cut view of the porter domain. The yellow circle indicates the closed pore-like structure comprising three central α-helices (depicted as a ribbon model with dense color), which was postulated to be a part of the putative substrate translocation channel during the early stages, however, it was not the case. The central α-helix of extrusion monomer is inclined 15° toward binding monomer more than the other two α-helices and, as a result, blocks the exit from the drug binding site. Bound minocycline (cyan), doxorubicin (orange), rifampicin (magenta), and erythromycin (yellow) overlap in the space-filling model.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4403515&req=5

Figure 5: Intramolecular water-accessible channels in the AcrB trimer (Nakashima et al., 2011). The channels are shown as colored solid surfaces, as calculated using the CAVER program (Medek et al., 2007). The proximal pocket, distal pocket, entrances and funnel-like exit are depicted in green, pink, gray, and yellow, respectively. The channel apertures at the entrance and exit are depicted in black. (1) Inner membrane entrance (Murakami et al., 2006), (2) periplasmic entrance (Seeger et al., 2006), (3) central cavity entrance (Nakashima et al., 2011). (A) Side view. The channels in the access monomer behind the figure have been omitted. The central cavity and central hole are depicted as dotted lines. (B) Horizontal cut view of the porter domain. The yellow circle indicates the closed pore-like structure comprising three central α-helices (depicted as a ribbon model with dense color), which was postulated to be a part of the putative substrate translocation channel during the early stages, however, it was not the case. The central α-helix of extrusion monomer is inclined 15° toward binding monomer more than the other two α-helices and, as a result, blocks the exit from the drug binding site. Bound minocycline (cyan), doxorubicin (orange), rifampicin (magenta), and erythromycin (yellow) overlap in the space-filling model.
Mentions: Three monomers of AcrB are tightly interacted to form a jelly-fish or mushroom-like structure (Figure 4A). Long hairpins are deeply inserted into the next monomers to form the shape that is likened to the figure called “it takes three to tango” (Lomovslaya et al., 2002). Three PN1 subdomains form a core for the headpiece and the three central α-helices form a pore-like structure at the center of the trimer (Figure 4B). The funnel-like 30 Å opening at the top of the TolC-docking domain is the same size as the bottom of the TolC channel (Koronakis et al., 2000). In the transmembrane domain, there is a central hole of 30 Å diameter surrounded by three 12-α-helix bundles (Figure 4C). The central hole is not a water-filled channel: it is filled with a phospholipid bilayer (Nakashima et al., 2013). There is a central cavity on the putative surface of the phospholipid bilayer in the central hole and below the closed pore-like structure (Figure 5A). The central cavity comprises three windows, named vestibules, to the surface of the inner membrane (Figures 5, 6). Initially, this central cavity was identified as a substrate binding site, at which the substrates are taken up through vestibules from the outer leaflet of the phospholipid bilayer membrane, bound to the central cavity, transferred through the central pore when it is open, and ultimately extruded from the funnel-like exit at the top of the AcrB trimer into the TolC channel (Murakami et al., 2002). Although the substrate-binding structures of the three-fold symmetry crystals in the central cavity were reported in 2003 (Yu et al., 2003), subsequent studies have not been able to identify significant electron densities of bound drugs in the central cavity [Pos et al. (2004); Murakami et al. (unpublished observation)] of the symmetric crystal.

Bottom Line: Multidrug recognition is based on a multisite drug-binding mechanism, in which two voluminous multidrug-binding pockets in cell membrane exporters recognize a wide range of substrates as a result of permutations at numerous binding sites that are specific for the partial structures of substrate molecules.Substrates are transported through dual multidrug-binding pockets via the peristaltic motion of the substrate translocation channel.Although there are no clinically available inhibitors of bacterial multidrug exporters, efforts to develop inhibitors based on structural information are underway.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Cell Membrane Structural Biology, Institute of Scientific and Industrial Research, Osaka University Ibaraki, Japan.

ABSTRACT
Bacterial multidrug exporters are intrinsic membrane transporters that act as cellular self-defense mechanism. The most notable characteristics of multidrug exporters is that they export a wide range of drugs and toxic compounds. The overexpression of these exporters causes multidrug resistance. Multidrug-resistant pathogens have become a serious problem in modern chemotherapy. Over the past decade, investigations into the structure of bacterial multidrug exporters have revealed the multidrug recognition and export mechanisms. In this review, we primarily discuss RND-type multidrug exporters particularly AcrAB-TolC, major drug exporter in Gram-negative bacteria. RND-type drug exporters are tripartite complexes comprising a cell membrane transporter, an outer membrane channel and an adaptor protein. Cell membrane transporters and outer membrane channels are homo-trimers; however, there is no consensus on the number of adaptor proteins in these tripartite complexes. The three monomers of a cell membrane transporter have varying conformations (access, binding, and extrusion) during transport. Drugs are exported following an ordered conformational change in these three monomers, through a functional rotation mechanism coupled with the proton relay cycle in ion pairs, which is driven by proton translocation. Multidrug recognition is based on a multisite drug-binding mechanism, in which two voluminous multidrug-binding pockets in cell membrane exporters recognize a wide range of substrates as a result of permutations at numerous binding sites that are specific for the partial structures of substrate molecules. The voluminous multidrug-binding pocket may have numerous binding sites even for a single substrate, suggesting that substrates may move between binding sites during transport, an idea named as multisite-drug-oscillation hypothesis. This hypothesis is consistent with the apparently broad substrate specificity of cell membrane exporters and their highly efficient ejection of drugs from the cell. Substrates are transported through dual multidrug-binding pockets via the peristaltic motion of the substrate translocation channel. Although there are no clinically available inhibitors of bacterial multidrug exporters, efforts to develop inhibitors based on structural information are underway.

No MeSH data available.


Related in: MedlinePlus