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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 analyses of the interaction of MtrC, MtrD, and MtrE. 200 μm NT-MtrC was titrated into (A) 15 μm MtrE and (B) 12 μm MtrD; and in (C) 15 μm MtrE was titrated into 12 μm MtrD in a VP-ITC microcalorimeter and the heat exchange determined at 25 °C. In each case, 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 interactants. Non-linear regression fitting of the data (shown as a solid line through the data points in the lower panel) to a monophasic one-site model yielded the following thermodynamic parameters for the interaction: the interaction of Nt-MtrC with MtrE was characterized by a Ka, ΔH and ΔS of 1.0 (±0.50) × 105 m−1, −6836 (±3575) cal·mol−1 and −0.01 cal·mol−1·K−1; while the interaction of Nt-MtrC with MtrD was characterized by a Ka, ΔH, and ΔS of 1.2 (±1.0) × 106 m−1, 1412 (±318) cal·mol−1 and 32.5 cal·mol−1·K−1.
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Figure 2: ITC analyses of the interaction of MtrC, MtrD, and MtrE. 200 μm NT-MtrC was titrated into (A) 15 μm MtrE and (B) 12 μm MtrD; and in (C) 15 μm MtrE was titrated into 12 μm MtrD in a VP-ITC microcalorimeter and the heat exchange determined at 25 °C. In each case, 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 interactants. Non-linear regression fitting of the data (shown as a solid line through the data points in the lower panel) to a monophasic one-site model yielded the following thermodynamic parameters for the interaction: the interaction of Nt-MtrC with MtrE was characterized by a Ka, ΔH and ΔS of 1.0 (±0.50) × 105 m−1, −6836 (±3575) cal·mol−1 and −0.01 cal·mol−1·K−1; while the interaction of Nt-MtrC with MtrD was characterized by a Ka, ΔH, and ΔS of 1.2 (±1.0) × 106 m−1, 1412 (±318) cal·mol−1 and 32.5 cal·mol−1·K−1.

Mentions: Microcalorimetry was used to assess the affinities of these interactions. When NT-MtrC was titrated into MtrE a weak exothermic interaction, with a Kd of 9.8 (±4.9) μm, was detected (Fig. 2A), which was characterized by a change in enthalpy (ΔH) of −6.8 (±3.58) kcal·mol−1 and entropy (ΔS) of −0.01 cal·mol−1·K−1. In contrast, when NT-MtrC was titrated into MtrD, a strong endothermic reaction, with a Kd of 0.8 (±0.70) μm, was detected (Fig. 2B), which was characterized by a ΔH of 1.4 (±0.32) kcal·mol−1 and ΔS of 32.5 cal·mol−1·K−1. In accord with our pulldown experiments that suggested that MtrD interacts weakly with MtrE, no appreciable interaction was detected by ITC (Fig. 2C). Our ITC measurements indicated that MtrE and MtrD have similar affinities for MtrC to that of TolC and AcrB for AcrA (7). In each case it is notable that the OMP binds the MFP with an affinity that is about an order-of-magnitude less than the IMP binds the MFP. In the case of TolC and AcrA this might have arisen because TolC needs to interact with a number of different transport systems. However, our studies established MtrC binds MtrE with a lower affinity than MtrD, suggesting that this is an intrinsic feature of such pumps, which might reflect a need to load and unload the OMP.


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 analyses of the interaction of MtrC, MtrD, and MtrE. 200 μm NT-MtrC was titrated into (A) 15 μm MtrE and (B) 12 μm MtrD; and in (C) 15 μm MtrE was titrated into 12 μm MtrD in a VP-ITC microcalorimeter and the heat exchange determined at 25 °C. In each case, 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 interactants. Non-linear regression fitting of the data (shown as a solid line through the data points in the lower panel) to a monophasic one-site model yielded the following thermodynamic parameters for the interaction: the interaction of Nt-MtrC with MtrE was characterized by a Ka, ΔH and ΔS of 1.0 (±0.50) × 105 m−1, −6836 (±3575) cal·mol−1 and −0.01 cal·mol−1·K−1; while the interaction of Nt-MtrC with MtrD was characterized by a Ka, ΔH, and ΔS of 1.2 (±1.0) × 106 m−1, 1412 (±318) cal·mol−1 and 32.5 cal·mol−1·K−1.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3037662&req=5

Figure 2: ITC analyses of the interaction of MtrC, MtrD, and MtrE. 200 μm NT-MtrC was titrated into (A) 15 μm MtrE and (B) 12 μm MtrD; and in (C) 15 μm MtrE was titrated into 12 μm MtrD in a VP-ITC microcalorimeter and the heat exchange determined at 25 °C. In each case, 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 interactants. Non-linear regression fitting of the data (shown as a solid line through the data points in the lower panel) to a monophasic one-site model yielded the following thermodynamic parameters for the interaction: the interaction of Nt-MtrC with MtrE was characterized by a Ka, ΔH and ΔS of 1.0 (±0.50) × 105 m−1, −6836 (±3575) cal·mol−1 and −0.01 cal·mol−1·K−1; while the interaction of Nt-MtrC with MtrD was characterized by a Ka, ΔH, and ΔS of 1.2 (±1.0) × 106 m−1, 1412 (±318) cal·mol−1 and 32.5 cal·mol−1·K−1.
Mentions: Microcalorimetry was used to assess the affinities of these interactions. When NT-MtrC was titrated into MtrE a weak exothermic interaction, with a Kd of 9.8 (±4.9) μm, was detected (Fig. 2A), which was characterized by a change in enthalpy (ΔH) of −6.8 (±3.58) kcal·mol−1 and entropy (ΔS) of −0.01 cal·mol−1·K−1. In contrast, when NT-MtrC was titrated into MtrD, a strong endothermic reaction, with a Kd of 0.8 (±0.70) μm, was detected (Fig. 2B), which was characterized by a ΔH of 1.4 (±0.32) kcal·mol−1 and ΔS of 32.5 cal·mol−1·K−1. In accord with our pulldown experiments that suggested that MtrD interacts weakly with MtrE, no appreciable interaction was detected by ITC (Fig. 2C). Our ITC measurements indicated that MtrE and MtrD have similar affinities for MtrC to that of TolC and AcrB for AcrA (7). In each case it is notable that the OMP binds the MFP with an affinity that is about an order-of-magnitude less than the IMP binds the MFP. In the case of TolC and AcrA this might have arisen because TolC needs to interact with a number of different transport systems. However, our studies established MtrC binds MtrE with a lower affinity than MtrD, suggesting that this is an intrinsic feature of such pumps, which might reflect a need to load and unload the OMP.

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