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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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.
The authors declare no competing financial interests.
Unfolding transitions of wild-type and mutant OmpA in lipid bilayers measured by fluorescence spectroscopy. (PDF 96 kb)
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)
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)
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)
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)
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)
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)
Analysis of additional double-mutant cycles and m values in double-mutant cycles. (DOC 33 kb)
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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