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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

Cut view of the transmembrane channels of the binding monomer (A) (blue) with bound doxorubicin (orange) and that of the access monomer (B) (green) with bound rifampicin (magenta). The switch loop containing Phe617 at the tip is depicted using a red ribbon model.
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Figure 7: Cut view of the transmembrane channels of the binding monomer (A) (blue) with bound doxorubicin (orange) and that of the access monomer (B) (green) with bound rifampicin (magenta). The switch loop containing Phe617 at the tip is depicted using a red ribbon model.

Mentions: Following minocycline and doxorubicin-binding structures, the drug binding structures of AcrB bound to the high molecular mass drugs (HMMD) rifampicin and erythromycin were reported (Nakashima et al., 2011). Similar to the low molecular mass drugs (LMMD) minocycline and doxorubicin, one molecule of rifampicin or erythromycin bound to one AcrB trimer. However, rifampicin and erythromycin binding monomer was not a binding monomer but an access monomer (Figures 4A,B). The rifampicin- and erythromycin-binding pocket is located between PC1 and PC2 in the substrate translocation channel between the entrance(s) and the LMMD binding pocket (Figure 5). Thus, the HMMD-binding pocket is referred to as a proximal pocket, and the LMMD-binding pocket is referred to as a distal pocket. Shortly after the report of proximal drug binding (Nakashima et al., 2011; Eicher et al., 2012) independently reported the presence of the proximal pocket in the access monomer in which the doxorubicin dimer is bound. The proximal pocket in the access monomer is voluminous permitting typical multisite-binding of the HMMDs rifampicin and erythromycin (Figure 4B insert). However, in access monomer, the distal pocket is smaller than the proximal pocket. In contrast, in the binding monomer, the distal pocket expands and the proximal pocket shrinks. Both pockets are separated by a switch loop (Figure 7). The path under the switch loop is too narrow for the HMMDs to move into the distal pocket. The switch loop swings during the conformational change from the access stage to the binding stage. HMMDs could be transferred from the proximal pocket to the distal pocket through the swinging of the switch loop and proximal pocket shrinking, followed by distal pocket expansion during the transition from the access to the binding stages (Nakashima et al., 2011). The importance of switch loop flexibility in export is supported by the fact that when site-directed mutagenesis fixes the loop through the introduction of double proline residues into the loop (Nakashima et al., 2011) or a G616N mutation (Cha et al., 2014), the resultant mutants have completely lost or significantly decreased the drug export activity. The crystal structure revealed that a double proline mutation fixed the loop conformation at a state between the access and binding stages (Nakashima et al., 2011).


Structural basis of RND-type multidrug exporters.

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

Cut view of the transmembrane channels of the binding monomer (A) (blue) with bound doxorubicin (orange) and that of the access monomer (B) (green) with bound rifampicin (magenta). The switch loop containing Phe617 at the tip is depicted using a red ribbon model.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 7: Cut view of the transmembrane channels of the binding monomer (A) (blue) with bound doxorubicin (orange) and that of the access monomer (B) (green) with bound rifampicin (magenta). The switch loop containing Phe617 at the tip is depicted using a red ribbon model.
Mentions: Following minocycline and doxorubicin-binding structures, the drug binding structures of AcrB bound to the high molecular mass drugs (HMMD) rifampicin and erythromycin were reported (Nakashima et al., 2011). Similar to the low molecular mass drugs (LMMD) minocycline and doxorubicin, one molecule of rifampicin or erythromycin bound to one AcrB trimer. However, rifampicin and erythromycin binding monomer was not a binding monomer but an access monomer (Figures 4A,B). The rifampicin- and erythromycin-binding pocket is located between PC1 and PC2 in the substrate translocation channel between the entrance(s) and the LMMD binding pocket (Figure 5). Thus, the HMMD-binding pocket is referred to as a proximal pocket, and the LMMD-binding pocket is referred to as a distal pocket. Shortly after the report of proximal drug binding (Nakashima et al., 2011; Eicher et al., 2012) independently reported the presence of the proximal pocket in the access monomer in which the doxorubicin dimer is bound. The proximal pocket in the access monomer is voluminous permitting typical multisite-binding of the HMMDs rifampicin and erythromycin (Figure 4B insert). However, in access monomer, the distal pocket is smaller than the proximal pocket. In contrast, in the binding monomer, the distal pocket expands and the proximal pocket shrinks. Both pockets are separated by a switch loop (Figure 7). The path under the switch loop is too narrow for the HMMDs to move into the distal pocket. The switch loop swings during the conformational change from the access stage to the binding stage. HMMDs could be transferred from the proximal pocket to the distal pocket through the swinging of the switch loop and proximal pocket shrinking, followed by distal pocket expansion during the transition from the access to the binding stages (Nakashima et al., 2011). The importance of switch loop flexibility in export is supported by the fact that when site-directed mutagenesis fixes the loop through the introduction of double proline residues into the loop (Nakashima et al., 2011) or a G616N mutation (Cha et al., 2014), the resultant mutants have completely lost or significantly decreased the drug export activity. The crystal structure revealed that a double proline mutation fixed the loop conformation at a state between the access and binding stages (Nakashima et al., 2011).

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