Crystal structures of a double-barrelled fluoride ion channel

Abstract

To contend with hazards posed by environmental fluoride, microorganisms export this anion through F--specific ion channels of the Fluc family1–4. Since the recent discovery of Fluc channels, numerous idiosyncratic features of these proteins have been unearthed, including extreme selectivity for F- over Cl- and dual-topology dimeric assembly5–6. To understand the chemical basis for F- permeation and how the antiparallel subunits convene to form a F--selective pore, we solved crystal structures of two bacterial Fluc homologues in complex with three different monobody inhibitors, with and without F- present, to a maximum resolution of 2.1 Å. The structures reveal a surprising “double-barrelled” channel architecture in which two F- ion pathways span the membrane and the dual-topology arrangement includes a centrally coordinated cation, most likely Na+. F- selectivity is proposed to arise from the very narrow pores and an unusual anion coordination that exploits the quadrupolar edges of conserved phenylalanine rings.

The molecular basis of sodium-dependent fluoride export by the eukaryotic fluoride channel FEX

“…These experiments support the hypothesis that the Phe box residues contribute to fluoride resistance: the F159I mutant did not grow at 0.5 mM NaF, whereas F349I was less impaired, only showing sensitivity at 5 mM NaF that might be explained by this construct’s lower expression level ( Figure 4B, Supplementary Figure 8A ). This is consistent with previous observations in bacteria and yeast, where mutation of the F349 equivalent is less detrimental than mutation of the F159 equivalent( 13, 22 ). Mutations to the other Phe residues that line the permeation pathway, F167I, F191I, F373I, and F294I, all cause substantial fluoride sensitivity at 0.5 mM NaF ( Figure 4B ), establishing the pore-lining phenylalanines as a key component of fluoride export activity in vivo .…”

The molecular basis of sodium-dependent fluoride export by the eukaryotic fluoride channel FEX

“…These experiments support the hypothesis that the Phe box residues contribute to fluoride resistance: the F159I mutant did not grow at 0.5 mM NaF, whereas F349I was less impaired, only showing sensitivity at 5 mM NaF that might be explained by this construct’s lower expression level ( Figure 4B, Supplementary Figure 8A ). This is consistent with previous observations in bacteria and yeast, where mutation of the F349 equivalent is less detrimental than mutation of the F159 equivalent( 13, 22 ). Mutations to the other Phe residues that line the permeation pathway, F167I, F191I, F373I, and F294I, all cause substantial fluoride sensitivity at 0.5 mM NaF ( Figure 4B ), establishing the pore-lining phenylalanines as a key component of fluoride export activity in vivo .…”

Ion Permeation, Selectivity, and Electronic Polarization in Fluoride Channels

“…Within the “gap”, the anion-π pairs with F80s govern ( Fig. 3C ), consistent with the observation that F80 mutants are nonfunctional (10,16). Next, N41 and H106 stabilize and guide F − to S ext ( Fig.…”

“…S84 HBonds with the S cen –marching/leaving F − . The irreplaceable “Phe-box” (10,11,16) has been hypothesized to form edgewise anion-π pairs with F − (16), which was confirmed by our simulations ( Fig. 3C ).…”

Asymmetry-induced distinct mechanisms and the transporting role of sodium in bacterial fluoride channel Fluc

“…We propose that Fluc does not operate via a single mechanism, but instead employs two distinct yet complementary modes of action, one in each pore, arising from the antiparallel, structurally antisymmetric assembly of the homodimer. Our results show that Pore I conforms more closely to the Channsporter mechanism originally proposed by Stockbridge et al (17). During the simulations, this pore exhibited slower, single-ion translocation events and longer F -residence times near the intracellular entrance, where the strongest sodium-fluoride interactions occurred.…”

The molecular basis of sodium-dependent fluoride export by the eukaryotic fluoride channel FEX

“…These experiments support the hypothesis that the Phe box residues contribute to fluoride resistance: the F159I mutant did not grow at 0.5 mM NaF, whereas F349I was less impaired, only showing sensitivity at 5 mM NaF that might be explained by this construct’s lower expression level ( Figure 4B, Supplementary Figure 8A ). This is consistent with previous observations in bacteria and yeast, where mutation of the F349 equivalent is less detrimental than mutation of the F159 equivalent( 13, 22 ). Mutations to the other Phe residues that line the permeation pathway, F167I, F191I, F373I, and F294I, all cause substantial fluoride sensitivity at 0.5 mM NaF ( Figure 4B ), establishing the pore-lining phenylalanines as a key component of fluoride export activity in vivo .…”

Ion Permeation, Selectivity, and Electronic Polarization in Fluoride Channels

“…Within the “gap”, the anion-π pairs with F80s govern ( Fig. 3C ), consistent with the observation that F80 mutants are nonfunctional (10,16). Next, N41 and H106 stabilize and guide F − to S ext ( Fig.…”

“…S84 HBonds with the S cen –marching/leaving F − . The irreplaceable “Phe-box” (10,11,16) has been hypothesized to form edgewise anion-π pairs with F − (16), which was confirmed by our simulations ( Fig. 3C ).…”

Asymmetry-induced distinct mechanisms and the transporting role of sodium in bacterial fluoride channel Fluc

“…We propose that Fluc does not operate via a single mechanism, but instead employs two distinct yet complementary modes of action, one in each pore, arising from the antiparallel, structurally antisymmetric assembly of the homodimer. Our results show that Pore I conforms more closely to the Channsporter mechanism originally proposed by Stockbridge et al (17). During the simulations, this pore exhibited slower, single-ion translocation events and longer F -residence times near the intracellular entrance, where the strongest sodium-fluoride interactions occurred.…”

The molecular basis of sodium-dependent fluoride export by the eukaryotic fluoride channel FEX

“…These experiments support the hypothesis that the Phe box residues contribute to fluoride resistance: the F159I mutant did not grow at 0.5 mM NaF, whereas F349I was less impaired, only showing sensitivity at 5 mM NaF that might be explained by this construct’s lower expression level ( Figure 4B, Supplementary Figure 8A ). This is consistent with previous observations in bacteria and yeast, where mutation of the F349 equivalent is less detrimental than mutation of the F159 equivalent( 13, 22 ). Mutations to the other Phe residues that line the permeation pathway, F167I, F191I, F373I, and F294I, all cause substantial fluoride sensitivity at 0.5 mM NaF ( Figure 4B ), establishing the pore-lining phenylalanines as a key component of fluoride export activity in vivo .…”

Ion Permeation, Selectivity, and Electronic Polarization in Fluoride Channels

“…Within the “gap”, the anion-π pairs with F80s govern ( Fig. 3C ), consistent with the observation that F80 mutants are nonfunctional (10,16). Next, N41 and H106 stabilize and guide F − to S ext ( Fig.…”

“…S84 HBonds with the S cen –marching/leaving F − . The irreplaceable “Phe-box” (10,11,16) has been hypothesized to form edgewise anion-π pairs with F − (16), which was confirmed by our simulations ( Fig. 3C ).…”

Asymmetry-induced distinct mechanisms and the transporting role of sodium in bacterial fluoride channel Fluc

“…We propose that Fluc does not operate via a single mechanism, but instead employs two distinct yet complementary modes of action, one in each pore, arising from the antiparallel, structurally antisymmetric assembly of the homodimer. Our results show that Pore I conforms more closely to the Channsporter mechanism originally proposed by Stockbridge et al (17). During the simulations, this pore exhibited slower, single-ion translocation events and longer F -residence times near the intracellular entrance, where the strongest sodium-fluoride interactions occurred.…”