An Interfacial Sodium Ion is an Essential Structural Feature of Fluc Family Fluoride Channels

McIlwain, Benjamin; Martin, Kamirah; Hayter, Elizabeth A.; Stockbridge, Randy B.

doi:10.1016/j.jmb.2020.01.007

“…We propose that the densities observed in the crystal structure either represent partially occupied fluoride sites, or that the monobodies used as crystallization chaperones alter the electrostatic landscape in the pore, increasing ion occupancy. Indeed, in crystal structures of Fluc-Bpe with monobody occupying only one side of the channel, each pore contained only one fluoride density, rather than two, in the polar track [11].…”

Section: Proposed Mechanism Of Fluoride Permeationmentioning

confidence: 99%

“…Crystal structures of representative Fluc channels from Bordetella pertussis (Fluc-Bpe) and an Escherichia coli virulence plasmid (Fluc-Ec2) provide an opportunity to understand the molecular basis for anion permeation in the Flucs ( Stockbridge et al, 2015 ; McIlwain et al, 2018 ). The protein possesses two deep, aqueous vestibules with an electropositive character due to an absolutely conserved arginine sidechain and a deeply buried sodium ion at the center of the protein ( Turman and Stockbridge, 2017 ; McIlwain et al, 2020 ). The structures captured four electron densities assigned as fluoride ions, two in each pore, positioned near the center of the protein, at some distance from the vestibules.…”

Section: Introductionmentioning

confidence: 99%

The fluoride permeation pathway and anion recognition in Fluc family fluoride channels

McIlwain

¹

,

Gundepudi

²

,

Koff

³

et al. 2021

eLife

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Fluc family fluoride channels protect microbes against ambient environmental fluoride by undermining the cytoplasmic accumulation of this toxic halide. These proteins are structurally idiosyncratic, and thus the permeation pathway and mechanism have no analogy in other known ion channels. Although fluoride binding sites were identified in previous structural studies, it was not evident how these ions access aqueous solution, and the molecular determinants of anion recognition and selectivity have not been elucidated. Using x-ray crystallography, planar bilayer electrophysiology and liposome-based assays, we identify additional binding sites along the permeation pathway. We use this information to develop an oriented system for planar lipid bilayer electrophysiology and observe anion block at one of these sites, revealing insights into the mechanism of anion recognition. We propose a permeation mechanism involving alternating occupancy of anion binding sites that are fully assembled only as the substrate approaches.

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“…We propose that the densities observed in the crystal structure either represent partially occupied fluoride sites, or that the monobodies used as crystallization chaperones alter the electrostatic landscape in the pore, increasing ion occupancy. Indeed, in crystal structures of Fluc-Bpe with monobody occupying only one side of the channel, each pore contained only one fluoride density, rather than two, in the polar track [10].…”

Section: Proposed Mechanism Of Fluoride Permeationmentioning

confidence: 99%

“…Mutant channels were constructed using standard molecular biology techniques and verified by sequencing. Histidine-tagged Fluc-Bpe and Fluc-Ec2 were expressed in E. coli and purified via cobalt affinity chromatography according to published protocols [3,5,10]. The buffer for the final size exclusion step was 100 mM NaBr, 10 mM 2-[4-(2-hydroxyethyl)piperazin-1yl]ethanesulfonic acid (HEPES), pH 7 for crystallography applications, or 100 mM NaCl, 10 mM HEPES pH 7 for functional reconstitution.…”

Section: Protein Expression Purification and Reconstitutionmentioning

confidence: 99%

“…Crystal structures of representative Fluc channels from Bordetella pertussis (Fluc-Bpe) and an Escherichia coli virulence plasmid (Fluc-Ec2) provide an opportunity to understand the molecular basis for anion permeation in the Flucs [5,9]. The protein possesses two deep, aqueous vestibules with electropositive character due to an absolutely conserved arginine sidechain and a deeply buried sodium ion at the center of the protein [10]. These structures captured four electron densities assigned as fluoride ions, two in each pore, positioned near the center of the protein, some distance from the vestibules.…”

Section: Introductionmentioning

confidence: 99%

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The fluoride permeation pathway and anion recognition in Fluc family fluoride channels

McIlwain

¹

,

Gundepudi

²

,

Koff

³

et al. 2021

Preprint

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View full text Add to dashboard Cite

Fluc family fluoride channels protect microbes against ambient environmental fluoride by undermining the cytoplasmic accumulation of this toxic halide. These proteins are structurally idiosyncratic, and thus the permeation pathway and mechanism have no analogy in other known ion channels. Although fluoride binding sites were identified in previous structural studies, it was not evident how these ions access aqueous solution, and the molecular determinants of anion recognition and selectivity have not been elucidated. Using x-ray crystallography, planar bilayer electrophysiology and liposome-based assays, we identify additional binding sites along the permeation pathway. We use this information to develop an oriented system for planar lipid bilayer electrophysiology and observe anion block at one of these sites, revealing insights into the mechanism of anion recognition. We propose a permeation mechanism involving alternating occupancy of anion binding sites that are fully assembled only as the substrate approaches.

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Recent Developments in Understanding Fluoride Accumulation, Toxicity, and Tolerance Mechanisms in Plants: an Overview

Gadi

¹

,

Kumar

²

,

Goswami

³

et al. 2020

J Soil Sci Plant Nutr

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Excess fluoride (F − ) inhibits the growth and metabolism of several crop plants, while some have an innate ability for F − tolerance. The present study was undertaken to explain how plants tolerate F − toxicity and understand the efficacy of exogenous stress protectants and phytoremediation approaches for F − homeostasis and tolerance of crop plants. This review comprehends existing information available so far on the F − accumulation toxicity and tolerance mechanisms. A high level of F − inhibits plant growth, water and nutrition uptake, enzyme activity, and yield. This review concludes that plants tolerate through F − homeostasis (exclusion, chelating, and compartmentalization) and gene activation for enhanced pathways of antioxidants, hormones, osmolytes, stress proteins, transporters, and metabolites. Exogenously applied stress protectants like salicylic acid (SA), polyamines (PAs), melatonin (Mel), glycine betaine, calcium (Ca 2+ ), and nanoparticles are differentially effective in reducing F − accumulation and toxicity by regulating various pathways. Application of minerals minimizes the F − accumulation through changing pH of soil, chelate formation with F − , and permeability of the cell membrane. Although many of the physiological aspects (enzyme activities, F − accumulation and regulation) were studied earlier, however, an effort has been made for the first time to understand the genetic basis and role of stress protectants and mineral nutrition, regulating these physiological activities, and explore new integrated approaches related to F − mitigation by means of phytoremediation. This review covers the recent development in F − transporters, gene expression, and biochemical and hormonal levels which could be utilized in regulating F − toxicity through omics and transgenic approaches in the future.

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An Interfacial Sodium Ion is an Essential Structural Feature of Fluc Family Fluoride Channels

Cited by 18 publications

References 33 publications

The fluoride permeation pathway and anion recognition in Fluc family fluoride channels

The fluoride permeation pathway and anion recognition in Fluc family fluoride channels

The fluoride permeation pathway and anion recognition in Fluc family fluoride channels

Recent Developments in Understanding Fluoride Accumulation, Toxicity, and Tolerance Mechanisms in Plants: an Overview

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