Conformational Flexibility in the Multidrug Efflux System Protein AcrA

Mikolosko, Jonathan; Bobyk, Kostyantyn D.; Zgurskaya, Helen I.; Ghosh, Partho

doi:10.1016/j.str.2005.11.015

Cited by 254 publications

(320 citation statements)

References 41 publications

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“…In the simplest interpretation, our results suggest a single binding site on the TolC protomer, interacting with the ␣-helical hairpin of a single adapter molecule. We saw no evidence for interaction with residues facing the TolC monomer interface on helices H3/H4/ H7* with any of the cross-linking agents, but weaker secondary contact sites cannot be excluded especially as ITC data indicate concentration-dependent binding events (6), and physically there would appear to be space to accommodate up to three adaptor subunits per TolC monomer (18,20).…”

Section: Discussionmentioning

confidence: 70%

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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

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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

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Section: Discussionmentioning

confidence: 70%

“…These interactions can be detected by cross-linking in vivo and measured in vitro by isothermal calorimetry (ITC) (6). Adaptor monomers have an elongated modular structure comprising a ␤-barrel, a lipoyl domain, and a long ␣-helical hairpin that extends over four or five heptad repeats and projects into the periplasm (18)(19)(20).…”

mentioning

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

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“…The MFP AcrA is crucial for the functional assembly of the pump by bridging AcrB and TolC in the periplasmic space (8,9), comprising the membrane proximal domain, ␤-barrel domain, lipoyl domain, and ␣-hairpin domain (10,11). The ␣-hairpin domain is associated with the TolC ␣-barrel domain (7,(12)(13)(14).…”

mentioning

confidence: 99%

Assembly and Channel Opening of Outer Membrane Protein in Tripartite Drug Efflux Pumps of Gram-negative Bacteria

Moeller

Jun

et al. 2012

Journal of Biological Chemistry

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Background: Pseudomonas aeruginosa mainly achieves multidrug resistance by use of the MexAB-OprM pump. Results: We determined the crystal structure of MexA. Electron microscopy work using MexA and OprM reveals that MexA makes a tip-to-tip interaction with OprM. Conclusion:We suggest an assembly and channel opening model for the pump. Significance: This study provides a better understanding of multidrug resistance in Gram-negative bacteria.

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“…This complex recruits a TolC exit duct, which is anchored in the outer membrane (OM) and projects across the periplasm. The structures of all three drug efflux pump components are known: the trimeric TolC exit duct (5), the trimeric AcrB transporter (6,7), the partial adaptor AcrA (8), and the complete homologous Pseudomonas adaptor MexA (9,10). We have established a model of the assembled tripartite machinery on the basis of extensive site-specific cross-linking between the flexible, linearly arranged multidomain adaptor and its two cognate partner proteins (9,11).…”

mentioning

confidence: 99%

Structures of sequential open states in a symmetrical opening transition of the TolC exit duct

Pei

Hinchliffe²,

Symmons³

et al. 2011

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

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In bacterial drug resistance and virulence pumps, an inner membrane (IM) transporter and periplasmic adaptor recruit an outer membrane (OM) trimeric TolC exit duct that projects an α-helical tunnel across the periplasm. The TolC periplasmic entrance is closed by densely packed α-helical coiled coils, inner H7/H8, and outer H3/H4, constrained by a hydrogen bond network. On recruitment, these coiled coils must undergo transition to the open state. We present 2.9 Å resolution crystal structures of two sequential TolC open states in which the network is incrementally disrupted and channel conductances defined in lipid bilayers. Superimposition of TolC RS (370 pS) and TolC YFRS (1,000 pS) on the TolC WT closed state (80 pS) showed that in the initial open-state TolC RS , relaxation already causes approximately 14°twisting and expansion of helix H7 at the periplasmic tip, increasing interprotomer distances from 12.2 Å in TolC WT to 18.9 Å. However, in the crystal structure, the weakened Asp 374 pore constriction was maintained at the closed state 11.3 Å 2 .In the advanced open-state TolC YFRS , there was little further expansion at the tip, to interprotomer 21.3 Å, but substantial movement of inner and outer coiled coils dilated the pore constriction. In particular, upon abolition of the TolC YFRS intraprotomer Tyr 362 -Asp 153 link, a redirection of Tyr 362 and "bulge" in H3 allowed a simple movement outward of H8, establishing a 50.3 Å 2 opening. Root mean square deviations (rmsds) over the coiled coils of the three protomers of TolC RS and TolC YFRS illustrate that, whereas independent movement at the periplasmic tips may feature in the initial stages of opening, full dilation of the pore constriction is entirely symmetrical.antibiotic resistance | drug efflux | type I export | X-ray crystal structure

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Conformational Flexibility in the Multidrug Efflux System Protein AcrA

Cited by 254 publications

References 41 publications

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

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

Assembly and Channel Opening of Outer Membrane Protein in Tripartite Drug Efflux Pumps of Gram-negative Bacteria

Structures of sequential open states in a symmetrical opening transition of the TolC exit duct

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