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Opening of the outer membrane protein channel in tripartite efflux pumps is induced by interaction with the membrane fusion partner.

Janganan TK, Zhang L, Bavro VN, Matak-Vinkovic D, Barrera NP, Burton MF, Steel PG, Robinson CV, Borges-Walmsley MI, Walmsley AR - J. Biol. Chem. (2010)

Bottom Line: A mutational analysis identified residues Asn-198, Glu-434, and Gln-441, lining an intraprotomer groove on the surface of MtrE, to be important for pump function; mutation of these residues yielded cells that were sensitive to vancomycin.Pull-down assays and micro-calorimetry measurements indicated that this functional impairment is not due to the inability of MtrC to interact with the MtrE mutants; nor was it due to the MtrE mutants adopting an open conformation, because cells expressing these MtrE mutants alone are relatively insensitive to vancomycin.However, cells expressing the MtrE mutants with MtrC are sensitive to vancomycin, indicating that residues lining the intra-protomer groove control opening of the MtrE channel in response to binding of MtrC.

View Article: PubMed Central - PubMed

Affiliation: School of Biological and Biomedical Sciences, Durham University, Durham DH1 3LE, United Kingdom.

ABSTRACT
The multiple transferable resistance (MTR) pump, from Neisseria gonorrhoeae, is typical of the specialized machinery used to translocate drugs across the inner and outer membranes of Gram-negative bacteria. It consists of a tripartite complex composed of an inner-membrane transporter, MtrD, a periplasmic membrane fusion protein, MtrC, and an outer-membrane channel, MtrE. We have expressed the components of the pump in Escherichia coli and used the antibiotic vancomycin, which is too large to cross the outer-membrane by passive diffusion, to test for opening of the MtrE channel. Cells expressing MtrCDE are not susceptible to vancomycin, indicating that the channel is closed; but become susceptible to vancomycin in the presence of transported substrates, consistent with drug-induced opening of the MtrE channel. A mutational analysis identified residues Asn-198, Glu-434, and Gln-441, lining an intraprotomer groove on the surface of MtrE, to be important for pump function; mutation of these residues yielded cells that were sensitive to vancomycin. Pull-down assays and micro-calorimetry measurements indicated that this functional impairment is not due to the inability of MtrC to interact with the MtrE mutants; nor was it due to the MtrE mutants adopting an open conformation, because cells expressing these MtrE mutants alone are relatively insensitive to vancomycin. However, cells expressing the MtrE mutants with MtrC are sensitive to vancomycin, indicating that residues lining the intra-protomer groove control opening of the MtrE channel in response to binding of MtrC.

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ITC analysis of the interaction of the MtrC hairpin with MtrE and its E434K. 500 μm MtrC hairpin was titrated into (A) 15 μm MtrE and (B) 15 μm) MtrE E434K in a VP-ITC micro-calorimeter and the heat exchange determined at 25 °C. The upper panel shows the raw energy changes during the titration, while the lower panel represents the derived integrated total energy change as a function of the molar ratio (based on the molecular weight of the monomeric protein) of the interactant. In the case of wild-type MtrE, for which the titration was extended to a molar ratio of 2.5, the data were best-fitted to a two site model yielded the following thermodynamic parameters for the interaction: Ka1, Ka2, ΔH1, ΔH2, ΔS1, and ΔS2 of 4.6 (±0.61) × 105 m−1, 3.2 (±0.96) × 103 m−1, −1.2 (±1.31) × 104 cal·mol−1,−6.2 (±0.01) × 104 cal·mol−1, −11.4 cal·mol−1·K−1, and −190 cal·mol−1·K−1, respectively. In the case of the MtrE E434K derivative, the titration was restricted to a molar ratio of 1.5, and consequently it was only appropriate to fit the data to a one site model; the high-affinity site was characterized by a Ka, ΔH and ΔS of 8.2 (±4.8) × 106 m−1, −4115 (±311) cal·mol−1 and 17.8 cal·mol−1·K−1.
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Figure 6: ITC analysis of the interaction of the MtrC hairpin with MtrE and its E434K. 500 μm MtrC hairpin was titrated into (A) 15 μm MtrE and (B) 15 μm) MtrE E434K in a VP-ITC micro-calorimeter and the heat exchange determined at 25 °C. The upper panel shows the raw energy changes during the titration, while the lower panel represents the derived integrated total energy change as a function of the molar ratio (based on the molecular weight of the monomeric protein) of the interactant. In the case of wild-type MtrE, for which the titration was extended to a molar ratio of 2.5, the data were best-fitted to a two site model yielded the following thermodynamic parameters for the interaction: Ka1, Ka2, ΔH1, ΔH2, ΔS1, and ΔS2 of 4.6 (±0.61) × 105 m−1, 3.2 (±0.96) × 103 m−1, −1.2 (±1.31) × 104 cal·mol−1,−6.2 (±0.01) × 104 cal·mol−1, −11.4 cal·mol−1·K−1, and −190 cal·mol−1·K−1, respectively. In the case of the MtrE E434K derivative, the titration was restricted to a molar ratio of 1.5, and consequently it was only appropriate to fit the data to a one site model; the high-affinity site was characterized by a Ka, ΔH and ΔS of 8.2 (±4.8) × 106 m−1, −4115 (±311) cal·mol−1 and 17.8 cal·mol−1·K−1.

Mentions: Because mutation of the intra-protomer groove impaired pump function, we tested whether this arose due to a reduced interaction of MtrC with the MtrE derivatives. A pulldown experiment was undertaken using GST-tagged NT MtrC as the bait and His-tagged MtrE E434K as the prey; revealing that the interaction was not compromised and that MtrC was capable of pulling-down MtrE E434K (Fig. 5). In comparison to the wild-type MtrE control, we did not see any reduction in the intensity of the band corresponding to His-tagged MtrE E434K on a Western blot, suggesting that MtrE and its E434K derivative have at least similar affinities for MtrC. Furthermore, we sought to test if the MtrE E434K derivative interacted with the coiled-coil hairpin domain of MtrC. For this experiment, we made a construct to express the hairpin domain of MtrC, consisting of residues 103–183; the purified protein had a CD spectrum that indicated that it retained α-helical structure (data not shown). Using ITC, we established that the hairpin binds to both wild-type MtrE and its E434K derivative (Fig. 6). Analysis of the binding isotherms indicated that the hairpin binds to a site on MtrE that is characterized by a Kd, ΔH and ΔS of 2.2(±0.29) μm, −11 (±13.1) kcal·mol−1 and −11.6 cal·mol−1·K−1; while its binding to the E434K derivative was characterized by a Kd, ΔH and ΔS of 0.13 (±0.078)μm, −4.1 (±0.31) kcal·mol−1 and 17.8 cal·mol−1·K−1. Interestingly, these data indicate that MtrE E434K binds the hairpin of MtrC with an affinity that is one-to-two orders of magnitude greater than the wild-type protein binds the hairpin or NT MtrC (see 0.13 μm versus 2.2/9.8 μm), suggesting that this derivative forms a tight assembly with MtrCD.


Opening of the outer membrane protein channel in tripartite efflux pumps is induced by interaction with the membrane fusion partner.

Janganan TK, Zhang L, Bavro VN, Matak-Vinkovic D, Barrera NP, Burton MF, Steel PG, Robinson CV, Borges-Walmsley MI, Walmsley AR - J. Biol. Chem. (2010)

ITC analysis of the interaction of the MtrC hairpin with MtrE and its E434K. 500 μm MtrC hairpin was titrated into (A) 15 μm MtrE and (B) 15 μm) MtrE E434K in a VP-ITC micro-calorimeter and the heat exchange determined at 25 °C. The upper panel shows the raw energy changes during the titration, while the lower panel represents the derived integrated total energy change as a function of the molar ratio (based on the molecular weight of the monomeric protein) of the interactant. In the case of wild-type MtrE, for which the titration was extended to a molar ratio of 2.5, the data were best-fitted to a two site model yielded the following thermodynamic parameters for the interaction: Ka1, Ka2, ΔH1, ΔH2, ΔS1, and ΔS2 of 4.6 (±0.61) × 105 m−1, 3.2 (±0.96) × 103 m−1, −1.2 (±1.31) × 104 cal·mol−1,−6.2 (±0.01) × 104 cal·mol−1, −11.4 cal·mol−1·K−1, and −190 cal·mol−1·K−1, respectively. In the case of the MtrE E434K derivative, the titration was restricted to a molar ratio of 1.5, and consequently it was only appropriate to fit the data to a one site model; the high-affinity site was characterized by a Ka, ΔH and ΔS of 8.2 (±4.8) × 106 m−1, −4115 (±311) cal·mol−1 and 17.8 cal·mol−1·K−1.
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Related In: Results  -  Collection

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Figure 6: ITC analysis of the interaction of the MtrC hairpin with MtrE and its E434K. 500 μm MtrC hairpin was titrated into (A) 15 μm MtrE and (B) 15 μm) MtrE E434K in a VP-ITC micro-calorimeter and the heat exchange determined at 25 °C. The upper panel shows the raw energy changes during the titration, while the lower panel represents the derived integrated total energy change as a function of the molar ratio (based on the molecular weight of the monomeric protein) of the interactant. In the case of wild-type MtrE, for which the titration was extended to a molar ratio of 2.5, the data were best-fitted to a two site model yielded the following thermodynamic parameters for the interaction: Ka1, Ka2, ΔH1, ΔH2, ΔS1, and ΔS2 of 4.6 (±0.61) × 105 m−1, 3.2 (±0.96) × 103 m−1, −1.2 (±1.31) × 104 cal·mol−1,−6.2 (±0.01) × 104 cal·mol−1, −11.4 cal·mol−1·K−1, and −190 cal·mol−1·K−1, respectively. In the case of the MtrE E434K derivative, the titration was restricted to a molar ratio of 1.5, and consequently it was only appropriate to fit the data to a one site model; the high-affinity site was characterized by a Ka, ΔH and ΔS of 8.2 (±4.8) × 106 m−1, −4115 (±311) cal·mol−1 and 17.8 cal·mol−1·K−1.
Mentions: Because mutation of the intra-protomer groove impaired pump function, we tested whether this arose due to a reduced interaction of MtrC with the MtrE derivatives. A pulldown experiment was undertaken using GST-tagged NT MtrC as the bait and His-tagged MtrE E434K as the prey; revealing that the interaction was not compromised and that MtrC was capable of pulling-down MtrE E434K (Fig. 5). In comparison to the wild-type MtrE control, we did not see any reduction in the intensity of the band corresponding to His-tagged MtrE E434K on a Western blot, suggesting that MtrE and its E434K derivative have at least similar affinities for MtrC. Furthermore, we sought to test if the MtrE E434K derivative interacted with the coiled-coil hairpin domain of MtrC. For this experiment, we made a construct to express the hairpin domain of MtrC, consisting of residues 103–183; the purified protein had a CD spectrum that indicated that it retained α-helical structure (data not shown). Using ITC, we established that the hairpin binds to both wild-type MtrE and its E434K derivative (Fig. 6). Analysis of the binding isotherms indicated that the hairpin binds to a site on MtrE that is characterized by a Kd, ΔH and ΔS of 2.2(±0.29) μm, −11 (±13.1) kcal·mol−1 and −11.6 cal·mol−1·K−1; while its binding to the E434K derivative was characterized by a Kd, ΔH and ΔS of 0.13 (±0.078)μm, −4.1 (±0.31) kcal·mol−1 and 17.8 cal·mol−1·K−1. Interestingly, these data indicate that MtrE E434K binds the hairpin of MtrC with an affinity that is one-to-two orders of magnitude greater than the wild-type protein binds the hairpin or NT MtrC (see 0.13 μm versus 2.2/9.8 μm), suggesting that this derivative forms a tight assembly with MtrCD.

Bottom Line: A mutational analysis identified residues Asn-198, Glu-434, and Gln-441, lining an intraprotomer groove on the surface of MtrE, to be important for pump function; mutation of these residues yielded cells that were sensitive to vancomycin.Pull-down assays and micro-calorimetry measurements indicated that this functional impairment is not due to the inability of MtrC to interact with the MtrE mutants; nor was it due to the MtrE mutants adopting an open conformation, because cells expressing these MtrE mutants alone are relatively insensitive to vancomycin.However, cells expressing the MtrE mutants with MtrC are sensitive to vancomycin, indicating that residues lining the intra-protomer groove control opening of the MtrE channel in response to binding of MtrC.

View Article: PubMed Central - PubMed

Affiliation: School of Biological and Biomedical Sciences, Durham University, Durham DH1 3LE, United Kingdom.

ABSTRACT
The multiple transferable resistance (MTR) pump, from Neisseria gonorrhoeae, is typical of the specialized machinery used to translocate drugs across the inner and outer membranes of Gram-negative bacteria. It consists of a tripartite complex composed of an inner-membrane transporter, MtrD, a periplasmic membrane fusion protein, MtrC, and an outer-membrane channel, MtrE. We have expressed the components of the pump in Escherichia coli and used the antibiotic vancomycin, which is too large to cross the outer-membrane by passive diffusion, to test for opening of the MtrE channel. Cells expressing MtrCDE are not susceptible to vancomycin, indicating that the channel is closed; but become susceptible to vancomycin in the presence of transported substrates, consistent with drug-induced opening of the MtrE channel. A mutational analysis identified residues Asn-198, Glu-434, and Gln-441, lining an intraprotomer groove on the surface of MtrE, to be important for pump function; mutation of these residues yielded cells that were sensitive to vancomycin. Pull-down assays and micro-calorimetry measurements indicated that this functional impairment is not due to the inability of MtrC to interact with the MtrE mutants; nor was it due to the MtrE mutants adopting an open conformation, because cells expressing these MtrE mutants alone are relatively insensitive to vancomycin. However, cells expressing the MtrE mutants with MtrC are sensitive to vancomycin, indicating that residues lining the intra-protomer groove control opening of the MtrE channel in response to binding of MtrC.

Show MeSH
Related in: MedlinePlus