Molecular Construction of a Multidrug Exporter System, AcrAB: Molecular Interaction between AcrA and AcrB, and Cleavage of the N-Terminal Signal Sequence of AcrA

Kawabe, Tetsuhiro; Fujihira, Erika; Yamaguchi, Akihito

doi:10.1093/oxfordjournals.jbchem.a022741

Cited by 26 publications

(20 citation statements)

References 0 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…However, preliminary experiments, using Western blot analysis with anti-AcrA and antiTolC antibodies did not reveal up-regulation of these proteins in the ⌬MKR strain (data not shown). Moreover, our ⌬MKR strain did not show enhanced resistance to SDS or ethidium bromide (data not shown), which are also pumped from the cell by the AcrABTolC system (38,39), although this result could be complicated by the regulatory mechanisms by which these compounds are sensed by the cell (40). Because erythromycin must enter and cross the inner membrane to gain access to ribosomes, a delay in this step could result in the efflux pumps being more effective at shedding the drug in the ⌬MKR strain, which would not necessarily require increased pump levels.…”

Section: Discussionmentioning

confidence: 80%

Revisiting the mechanism of macrolide-antibiotic resistance mediated by ribosomal protein L22

Moore

Sauer²

2008

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

Bacterial antibiotic resistance can occur by many mechanisms. An intriguing class of mutants is resistant to macrolide antibiotics even though these drugs still bind to their targets. For example, a 3-residue deletion (⌬MKR) in ribosomal protein L22 distorts a loop that forms a constriction in the ribosome exit tunnel, apparently allowing nascent-chain egress and translation in the presence of bound macrolides. Here, however, we demonstrate that ⌬MKR and wild-type ribosomes show comparable macrolide sensitivity in vitro. In Escherichia coli, we find that this mutation reduces antibiotic occupancy of the target site on ribosomes in a manner largely dependent on the AcrAB-TolC efflux system. We propose a model for antibiotic resistance in which ⌬MKR ribosomes alter the translation of specific proteins, possibly via changes in programmed stalling, and modify the cell envelope in a manner that lowers steady-state macrolide levels.ermC ͉ erythromycin ͉ ribosome ͉ TolC M any antibiotics inhibit bacterial protein synthesis. Understanding how microbes become antibiotic resistant is important both for developing effective treatment regimens and designing new therapeutics. Macrolides consist of a 14-to 16-member lactone ring with different appended sugars and comprise a key group of inhibitors of bacterial translation (1, 2). The inhibitory activity of macrolides, including erythromycin, depends on binding to a site near the polypeptide exit tunnel of the large ribosomal subunit (3, 4). Because macrolides do not bind to ribosomes with an occupied exit tunnel and cause the synthesis of 2-10 residue peptides in translation assays in vitro, it has been proposed that drug binding physically blocks elongation of nascent proteins beyond this size (5-7).Some macrolide-resistance mutations alter the ribosomal target site and prevent binding (4,8). Intriguingly, other mutations confer resistance despite the fact that macrolides still bind the mutant ribosome well (8-10). For example, deletion of the M 82 K 83 R 84 sequence in Escherichia coli ribosomal protein L22 (⌬MKR) allows growth in the presence of high levels of erythromycin and other macrolides (11-13). The same mutation makes Haemophilus influenzae resistant to numerous macrolides (14); different L22 mutations also confer macrolide resistance in other bacterial species (2). When binding has been measured, ribosomes with macrolideresistant alterations in L22 bind erythromycin with near wild-type affinity (8,10,12,15).In a crystal structure of the E. coli ribosome, the MKR sequence is part of an extended L22 loop, which together with a similar loop in protein L4 forms a narrow constriction in the exit tunnel (Fig. 1A) (16, 17). Cryo-EM studies initially revealed a widened exit tunnel in E. coli ⌬MKR ribosomes (18). This loop is also displaced to create an expanded tunnel in structures of ⌬MKR ribosomes from Thermus thermophilus and Haloarcula marismortui (4, 19). These results explain the altered chemical reactivity in E. coli ⌬MKR ribosomes of 23S-RNA bases (13). Together,...

show abstract

Section: Discussionmentioning

confidence: 80%

Revisiting the mechanism of macrolide-antibiotic resistance mediated by ribosomal protein L22

Moore

Sauer²

2008

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

show abstract

“…The His-tagged wild-type TolC was detected consistently in all samples, and no copurification of AcrA was observed in the negative controls without cross-linker or with the wild-type AcrA, which has no freely accessible cysteines (the sole natural cysteine is at the extreme N terminus, but it is lipid-modified and anchored in the inner membrane after removal of the signal sequence; ref. 28). AcrA-TolC SPDP cross-links were evident with AcrA-A 79 C, -D 87 C, -L 100 C, -R 104 C, -Q 112 C, and AcrA-E 118 C, but not the other variants.…”

Section: Resultsmentioning

confidence: 99%

A periplasmic coiled-coil interface underlying TolC recruitment and the assembly of bacterial drug efflux pumps

Lobedanz

Bokma²,

Symmons³

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

120

156

View full text Add to dashboard Cite

Bacteria such as Escherichia coli and Pseudomonas aeruginosa expel antibiotics and other inhibitors via tripartite multidrug efflux pumps spanning the inner and outer membranes and the intervening periplasmic space. A key event in pump assembly is the recruitment of an outer membrane-anchored TolC exit duct by the adaptor protein of a cognate inner membrane translocase, establishing a contiguous transenvelope efflux pore. We describe the underlying interaction of juxtaposed periplasmic exit duct and adaptor coiled-coils in the widespread RND-type pump TolC/AcrAB of E. coli, using in vivo cross-linking to map the extent of intermolecular contacts. Cross-linking of site-specific TolC cysteine variants to wild-type AcrA adaptor identified residues on the lower ␣-helical barrel domain of TolC, defining a contiguous cluster close to the entrance aperture of the exit duct. Reciprocally, site-specific cross-linking of AcrA cysteine variants to wild-type TolC identified the interaction surface on the adaptor within the N-terminal ␣-helix of the AcrA coiled-coil. The experimental data allowed a data-driven docking approach to model the interaction surface central to pump assembly. The lowest energy docked model satisfying all of the cross-link distance constraints places the adaptor at the intramolecular groove formed by the TolC entrance helices, aligning the adaptor coiled-coil with the exposed TolC outer helix. A key feature of this positioning is that it allows space for the proposed movement of the inner coil of TolC during transition to its open state.antibiotic resistance ͉ exit duct ͉ membrane proteins ͉ type I export

show abstract

“…AcrA is lipidated and then cleaved off at Cys25 by signal peptidase II, resulting in an N-terminal lipidated Cys25 residue that is anchored to the inner membrane during maturation (31). The lipidation and cleavage of AcrA are completely prevented when Cys25 is replaced with Ala (32). The resultant long AcrA molecule (AcrA L ) still possesses a transmembrane signal sequence and still maintains efflux activity (32).…”

mentioning

confidence: 99%

“…The lipidation and cleavage of AcrA are completely prevented when Cys25 is replaced with Ala (32). The resultant long AcrA molecule (AcrA L ) still possesses a transmembrane signal sequence and still maintains efflux activity (32).…”

mentioning

confidence: 99%

AcrB-AcrA Fusion Proteins That Act as Multidrug Efflux Transporters

et al. 2016

View full text Add to dashboard Cite

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.

show abstract

Molecular Construction of a Multidrug Exporter System, AcrAB: Molecular Interaction between AcrA and AcrB, and Cleavage of the N-Terminal Signal Sequence of AcrA

Cited by 26 publications

References 0 publications

Revisiting the mechanism of macrolide-antibiotic resistance mediated by ribosomal protein L22

Revisiting the mechanism of macrolide-antibiotic resistance mediated by ribosomal protein L22

A periplasmic coiled-coil interface underlying TolC recruitment and the assembly of bacterial drug efflux pumps

AcrB-AcrA Fusion Proteins That Act as Multidrug Efflux Transporters

Contact Info

Product

Resources

About