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Mechanism of single- and double-sided inhibition of dual topology fluoride channels by synthetic monobodies
Abstract
The Fluc family of proteins comprises small, electrodiffusive fluoride channels, which prevent accumulation of toxic F− ions in microorganisms. Recent crystal structures have confirmed their unusual architecture, in which a pair of antiparallel subunits convenes to form a dimer with a twofold symmetry axis parallel to the plane of the membrane. These structures have also revealed the interactions between Fluc channels and several different fibronectin domain monobodies that inhibit Fluc-mediated F− currents; in all structures, each channel binds to two monobodies symmetrically, one on either side of the membrane. However, these structures do not reveal the mechanism of monobody inhibition. Moreover, the results appear to diverge from a recent electrophysiological study indicating that monobody binding is negatively cooperative; that is, a bound monobody on one side of a Fluc channel decreases the affinity of an oppositely bound monobody by ∼10-fold. In this study, we reconcile these observations by probing the mechanism of monobody binding and its negative cooperativity using electrophysiological experiments in planar lipid bilayers. Our results indicate that monobody inhibition occurs via a pore-blocking mechanism and that negative cooperativity arises from electrostatic repulsion between the oppositely bound monobodies. A single glutamate residue, on a loop of the monobody that extends into the channel interior, is responsible for negatively cooperative binding. This glutamate side chain also confers voltage dependence and sensitivity to the concentration of trans-F− ion to monobody binding. Neutralization by mutation to glutamine abolishes these electrostatic effects. Monobodies that are amenable to cocrystallization with Fluc channels lack an analogous negatively charged side chain and bind independently to opposite sides of the channel. Thus, this work reveals the source of voltage dependence and negative cooperativity of monobody binding to Fluc channels along with the pore-blocking mechanism.
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Proteins encoded by the crcB genes are electrodiffusive F− channels.
“Electrophysiological characterization showed that the proteins encoded by the crcB genes are electrodiffusive F− channels (2, 3, 60–63), and they were renamed Fluc (Fluoride channel) in bacteria and FEX (Fluoride exporter) in eukaryotes.”
From Membrane exporters of fluoride ion (2021) · cites this paper as [62]
An inhibitor applied in sequence to the cis and trans sides of a single Fluc channel in a planar lipid bilayer blocks both sides of the channel with identical kinetics.
“Functional experiments support this observation: an inhibitor applied in sequence to the cis and trans sides of a single Fluc channel in a planar lipid bilayer blocks both sides of the channel with identical kinetics (60–62).”
From Membrane exporters of fluoride ion (2021) · cites this paper as [62]
Synthetic proteins that inhibit fluoride channels have been developed but have not been tested in biological systems, and no such natural regulatory mechanism has been identified.
“Although synthetic proteins that inhibit fluoride channels have been developed123–125, they have not been tested in biological systems, and no such natural regulatory mechanism has been identified.”
From The link between ancient microbial fluoride resistance mechanisms and bioengineering organofluorine degradation or synthesis (2024) · cites this paper as [124]
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Cites
Comparative protein structure modeling using MODELLER
Webb, B., & Sali, A. (2016) · Current Protocols in Protein Science · doi:10.1002/cpps.20 · Cited by 730
Crystal structures of a double-barrelled fluoride ion channel
Stockbridge, R. B., Kolmakova-Partensky, L., Shane, T., Koide, A., Koide, S., Miller, C., & Newstead, S. (2015) · Nature · doi:10.1038/nature14981 · Cited by 119
Self-citationBacterial fluoride resistance, Fluc channels, and the weak acid accumulation effect
Ji, C., Stockbridge, R. B., & Miller, C. (2014) · Journal of General Physiology · doi:10.1085/jgp.201411243 · Cited by 97
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Cited by
Sorted by citations
Stockbridge, R. B., & Wackett, L. P. (2024) · Nature Communications · doi:10.1038/s41467-024-49018-1 · Cited by 80
Self-citationMembrane exporters of fluoride ion
McIlwain, B. C., Ruprecht, M. T., & Stockbridge, R. B. (2021) · Annual Review of Biochemistry · doi:10.1146/annurev-biochem-071520-112507 · Cited by 69
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Metadata
- Authors
- Turman, D. L., & Stockbridge, R. B.
- Venue
- Journal of General Physiology
- Publisher
- Rockefeller University Press
- Published
- 2017-03-03
- Volume
- 149
- Issue
- 4
- Pages
- 511-522
- ISSN
- 0022-1295
- 1540-7748
- PMC
- PMC5379923
- Cited by
- 15
- References
- 3
- Language
- en
- Funders
- National Institutes of Health (R00-GM111767)
- Howard Hughes Medical Institute
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