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The C-terminal domain of AcrA is essential for the assembly and function of the multidrug efflux pump AcrAB-TolC.
J Bacteriol. 2009 Jul; 191(13):4365-71.JB

Abstract

Periplasmic membrane fusion proteins (MFPs) are essential components of multidrug efflux pumps and type I protein secretion systems of gram-negative bacteria. Located in the periplasm, MFPs function by creating a physical link between inner membrane transporters and outer membrane channels. The most conserved sequence of MFPs is located in their distal C-terminal domain. However, neither the structure nor the function of this domain is known. In this study, we investigated the structural and functional role of the C-terminal domain of Escherichia coli AcrA, a periplasmic component of the multidrug efflux pump AcrAB-TolC. Using trypsin proteolysis, we identified the proteolytically labile sites in the C-terminal domain (amino acid residues 315 to 397) of AcrA in vitro. We next used these sites as a map to evaluate the structural integrity of this domain of AcrA inside the periplasm. We found that the C-terminal domain of AcrA is protected from trypsin when the tripartite efflux pump AcrAB-TolC is assembled. In contrast, this domain remains proteolytically labile in cells producing only one of the AcrB or TolC components of the complex. Site-directed mutagenesis of 12 highly conserved amino acid residues of the C-terminal domain of AcrA showed that a single G363C substitution dramatically impairs the multidrug efflux activity of AcrAB-TolC. The G363C mutant interacts with both AcrB and TolC but fails to properly assemble into a functional complex. We conclude that the C-terminal domain of AcrA plays an important role in the assembly and function of AcrAB-TolC efflux pump.

Authors+Show Affiliations

Department of Chemistry and Biochemistry, University of Oklahoma, Norman, Oklahoma 73019, USA.No affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article

Language

eng

PubMed ID

19411330

Citation

Ge, Qiang, et al. "The C-terminal Domain of AcrA Is Essential for the Assembly and Function of the Multidrug Efflux Pump AcrAB-TolC." Journal of Bacteriology, vol. 191, no. 13, 2009, pp. 4365-71.
Ge Q, Yamada Y, Zgurskaya H. The C-terminal domain of AcrA is essential for the assembly and function of the multidrug efflux pump AcrAB-TolC. J Bacteriol. 2009;191(13):4365-71.
Ge, Q., Yamada, Y., & Zgurskaya, H. (2009). The C-terminal domain of AcrA is essential for the assembly and function of the multidrug efflux pump AcrAB-TolC. Journal of Bacteriology, 191(13), 4365-71. https://doi.org/10.1128/JB.00204-09
Ge Q, Yamada Y, Zgurskaya H. The C-terminal Domain of AcrA Is Essential for the Assembly and Function of the Multidrug Efflux Pump AcrAB-TolC. J Bacteriol. 2009;191(13):4365-71. PubMed PMID: 19411330.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - The C-terminal domain of AcrA is essential for the assembly and function of the multidrug efflux pump AcrAB-TolC. AU - Ge,Qiang, AU - Yamada,Yoichi, AU - Zgurskaya,Helen, Y1 - 2009/05/01/ PY - 2009/5/5/entrez PY - 2009/5/5/pubmed PY - 2009/7/1/medline SP - 4365 EP - 71 JF - Journal of bacteriology JO - J. Bacteriol. VL - 191 IS - 13 N2 - Periplasmic membrane fusion proteins (MFPs) are essential components of multidrug efflux pumps and type I protein secretion systems of gram-negative bacteria. Located in the periplasm, MFPs function by creating a physical link between inner membrane transporters and outer membrane channels. The most conserved sequence of MFPs is located in their distal C-terminal domain. However, neither the structure nor the function of this domain is known. In this study, we investigated the structural and functional role of the C-terminal domain of Escherichia coli AcrA, a periplasmic component of the multidrug efflux pump AcrAB-TolC. Using trypsin proteolysis, we identified the proteolytically labile sites in the C-terminal domain (amino acid residues 315 to 397) of AcrA in vitro. We next used these sites as a map to evaluate the structural integrity of this domain of AcrA inside the periplasm. We found that the C-terminal domain of AcrA is protected from trypsin when the tripartite efflux pump AcrAB-TolC is assembled. In contrast, this domain remains proteolytically labile in cells producing only one of the AcrB or TolC components of the complex. Site-directed mutagenesis of 12 highly conserved amino acid residues of the C-terminal domain of AcrA showed that a single G363C substitution dramatically impairs the multidrug efflux activity of AcrAB-TolC. The G363C mutant interacts with both AcrB and TolC but fails to properly assemble into a functional complex. We conclude that the C-terminal domain of AcrA plays an important role in the assembly and function of AcrAB-TolC efflux pump. SN - 1098-5530 UR - https://www.unboundmedicine.com/medline/citation/19411330/The_C_terminal_domain_of_AcrA_is_essential_for_the_assembly_and_function_of_the_multidrug_efflux_pump_AcrAB_TolC_ L2 - http://jb.asm.org/cgi/pmidlookup?view=long&pmid=19411330 DB - PRIME DP - Unbound Medicine ER -