Electrostatic couplings in OmpA ion-channel gating suggest a mechanism for pore opening

Abstract

The molecular forces that drive structural transitions between the open and closed states of channels and transporters are not well understood. The gate of the OmpA channel is formed by the central Glu52-Arg138 salt bridge, which can open to form alternate ion pairs with Lys82 and Glu128. To gain deeper insight into the channel-opening mechanism, we measured interaction energies between the relevant side chains by double-mutant cycle analysis and correlated these with the channel activities of corresponding point mutants. The closed central salt bridge has a strong interaction energy of −5.6 kcal mol−1, which can be broken by forming the open-state salt bridge Glu52-Lys82 (ΔΔGInter = −3.5 kcal mol−1) and a weak interaction between Arg138 and Glu128 (ΔΔGInter = −0.6 kcal mol−1). A covalent disulfide bond in place of the central salt bridge completely blocks the channel. Growth assays indicate that this gating mechanism could physiologically contribute to the osmoprotection of Escherichia coli cells from environmental stress.

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Figure 1: Configuration of side chains in the gate region of OmpA, based on the high-resolution crystal structure of its transmembrane domain.
Figure 2: Comparison of the structural and folding properties of wild-type and several mutant OmpAs in diC16:1PC:C16:0C18:1PG (89.5:10.5) bilayers at 37.5 °C, pH 9.2.
Figure 3: Double-mutant cycles for estimating the interaction energies, ΔΔGInter, between the charged side chains in the gating region of the OmpA channel.
Figure 4: Representative single-channel recordings of the small conductance states of wild-type and mutant OmpAs in planar lipid bilayers at +100 mV.
Figure 5: Statistical analysis of the single-channel recordings of wild-type and mutant OmpAs in planar bilayers.
Figure 6: Reversible blockage of channel properties by disulfide formation in place of the central salt bridge of OmpA.
Figure 7: Correlations between pair-wise side chain interactions, single-channel openings and conformational dynamics measured by NMR relaxation suggest a self-consistent model for the opening of the OmpA channel.

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Acknowledgements

We thank D. Reinhart for technical assistance, M. Kim and D. Cafiso (University of Virginia) for sharing the BL21(DE3) ([ΔlamB ompF::Tn5 ΔompA ΔompC]) strain, and the late R. Kadner (University of Virginia) and R. Kaback (University of California Los Angeles) for helpful suggestions. This work was supported by US National Institutes of Health grant GM51329.

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H.H. performed all experiments, H.H. and L.K.T. designed the experiments and wrote the manuscript, and G.S. participated in the electrophysiological experiments.

Corresponding author

Correspondence to Lukas K Tamm.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Fig. 1

Unfolding transitions of wild-type and mutant OmpA in lipid bilayers measured by fluorescence spectroscopy. (PDF 96 kb)

Supplementary Fig. 2

Comparison of the SDS-PAGE patterns for the boiled samples of wild type, E52C R138C mutant, and the transmembrane domains of wild type and E52C R138C mutants in the presence and absence of 1% βME. (PDF 615 kb)

Supplementary Fig. 3

SDS-PAGE of the outer-membrane fractions of E. coli BL21(DE3) mutant cells expressing wild-type and E52C R138C mutant OmpA with and without added 1% NaCl in the growth media. (PDF 894 kb)

Supplementary Fig. 4

Comparison of the growth behavior of E. coli BL21(DE3) mutant cells expressing the plasmid-coded OmpF in LB media with different NaCl concentrations. (PDF 331 kb)

Supplementary Fig. 5

Growth of E. coli BL21(DE3) mutant cells expressing the plasmid-coded E52C K82C mutant in the presence and absence of 0.05% βME in LB media with no added NaCl. (PDF 320 kb)

Supplementary Fig. 6

Effects of sucrose on growth behavior of E. coli BL21(DE3) mutant cells expressing the plasmid-coded wild-type OmpA and E52C R138C mutant in the presence and absence of 0.05% βME. (PDF 182 kb)

Supplementary Fig. 7

Additivity of m-value effects in double-mutant cycles for probing the interaction energies between the side chains in the gating region of the OmpA channel. (PDF 304 kb)

Supplementary Results

Analysis of additional double-mutant cycles and m values in double-mutant cycles. (DOC 33 kb)

Supplementary Methods

Supplementary biochemical, spectroscopic and analytical methods. (DOC 50 kb)

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Hong, H., Szabo, G. & Tamm, L. Electrostatic couplings in OmpA ion-channel gating suggest a mechanism for pore opening. Nat Chem Biol 2, 627–635 (2006). https://doi.org/10.1038/nchembio827

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