A CLC-type F-/H+ antiporter in ion-swapped conformations

Sequence alignments indicate that both 1289 and 1290 possess the characteristic fluoride-binding motifs observed in structures of the CLCF from Enterococcus casseliflavus.

“Sequence alignments indicate that both 1289 and 1290 possess the characteristic fluoride-binding motifs observed in structures of the CLCF from Enterococcus casseliflavus (26) (Fig. S5)”

Sequence alignments show that most of the fluoride-binding residues identified in the structure of the Enterococcus casseliflavus CLCF are conserved in 1290, except T320.

“Sequence alignments show that most of the fluoride-binding residues identified in the structure of the Enterococcus casseliflavus CLCF (26) are conserved in 1290, except T320 (E. casseliflavus numbering) (Fig. S5).”

In the E. casseliflavus protein, the T320A mutation inhibited transport.

“In the E. casseliflavus protein, the T320A mutation inhibited transport (26)”

CLCFs assemble as homodimers.

“Like other CLC proteins, CLCFs assemble as homodimers(51, 52).”

Fluoride-specific CLCFs can be identified by three signature sequences: GNNLI/GMGLI in the N-terminal domain, GREGT/V at the transport machinery, and GEVTP in the C-terminal domain.

“Fluoride-specific CLCFs can be identified based on three signature sequences: the GNNLI/GMGLI in the N-terminal domain that defines ion selectivity, GREGT/V at the heart of the transport machinery, and the GEVTP sequence in the C-terminal domain that contributes fluoride-binding residues77–79.”

F− transport is abolished in neutral mutants E118Q and E118A.

“In accord with the proposal that electrostatic repulsion by the E118 carboxylate hastens F− along the permeation pathway, F− transport is abolished in neutral mutants E118Q and E118A – because the F− binding affinity is over an order of magnitude higher in these mutants compared with wildtype(50, 52).”

M79 contributes to F− coordination via its terminal methyl group, which is polarized and rendered weakly electropositive by the adjacent electron-withdrawing sulfur.

“A methionine, M79, also contributes to F− coordination via the terminal methyl group of the sidechain, which is polarized and rendered weakly electropositive by the adjacent electron-withdrawing sulfur.”

Upon protonation, the neutral glutamate is proposed to swing down, following a hydrophobic pathway and bypassing the electropositive F− binding sites, to access the intracellular vestibule and release the bound proton to the cytoplasm.

“Upon protonation, the now-neutral glutamate is proposed to swing down, following a hydrophobic pathway and bypassing the electropositive F− binding sites (stage 2), so that it extends downward to access the intracellular vestibule, where it can release the bound proton to the cytoplasm (stage 3).”

The α-helices are tilted with respect to the membrane, defining electropositive internal and external aqueous vestibules that are 6-8 Å deep.

“In accordance with their role in anion transport, the a-helices are tilted with respect to the membrane, defining electropositive internal and external aqueous vestibules that are 6–8 Å deep (Figure 3c).”

The CLCF transport cycle proposed by Last, Miller, and colleagues is based on competition between F− and the negatively charged carboxylate of E118 for the binding sites Fcen and Fext.

“The CLCF transport cycle proposed by Last, Miller, and colleagues(52), is based on competition between F− and the negatively charged carboxylate of E118 for the binding sites Fcen and Fext (Figure 4).”

The Fext ion is coordinated by the sidechain of T320 and backbone amides from G116-G119, contributed by a helical break.

“This ion is coordinated by the sidechain of T320 along with backbone amides from consecutive residues, G116-G119, contributed by a helical break.”

In the wildtype CLCF structure, F− ions occupy both the Fcen and the dehydrated Fext site.

“In the structure of wildtype CLCF, F− ions occupy both the Fcen and the dehydrated Fext site.”

F− binding affinity is over an order of magnitude higher in E118Q and E118A mutants compared with wildtype.

“In accord with the proposal that electrostatic repulsion by the E118 carboxylate hastens F− along the permeation pathway, F− transport is abolished in neutral mutants E118Q and E118A – because the F− binding affinity is over an order of magnitude higher in these mutants compared with wildtype(50, 52).”

A conformationally swapped state, with E118 exposed to the internal solution, was captured in the crystal structure of a mutant transporter, V319G.

“Such a conformationally swapped state, with E118 exposed to the internal solution, was captured in the crystal structure of a mutant transporter, V319G (52).”

The Fext site is entirely dehydrated.

“The second F− binding site observed in the crystal structure, Fext, is entirely dehydrated.”

The crystal structures exhibit electron densities in two positions along the ion permeation pathway, termed the central (Fcen) and external (Fex) anion binding sites.

“The crystal structures exhibit electron densities in two positions along the ion permeation pathway, termed the central (Fcen) and external (Fex) anion binding sites (52) (Figure 3d).”

E118 is a critical proton carrier that faces the external solution in the wildtype E. casseliflavus CLCF structure.

“In addition to the fluoride-coordinating residues, a final critical component of the transport machinery is the proton carrier, E118, which faces the external solution in the structure of the wildtype E. casseliflavus CLCF.”

The Fcen site sits at the apex of the cytoplasmic vestibule, exposed to water and coordinated by protein sidechains in a manner reminiscent of other CLCs.

“The first of the fluoride binding sites, Fcen, sits at the apex of the cytoplasmic vestibule, where it is exposed to water and coordinated by protein sidechains in a manner reminiscent of other CLCs.”

In stage 1 of the transport cycle, the sidechain of E118 is in the 'up' position, where it can obtain a proton from the extracellular milieu.

“In stage 1 of the transport cycle, the sidechain of the gating glutamate E118 is in the ‘up’ position, where it can obtain a proton from the extracellular milieu.”

Each monomer of the CLCF homologue from Enterococcus casseliflavus possesses 14 transmembrane α-helices comprising two structurally homologous 7-helix domains related by internal inverted symmetry.

“X-ray crystal structures of a CLCF homologue from Enterococcus casseliflavus(52) show that each monomer possesses 14 transmembrane a-helices comprising two structurally homologous 7-helix domains that are related to one another by internal inverted symmetry (Figure 3a, 3b).”

In the absence of F−, E118Q and E118A mutants support Cl− efflux, albeit uncoupled to H+ exchange.

“In the absence of F−, these mutants support Cl− efflux, albeit uncoupled to H+ exchange(52).”

Breaks in these helices position the positive dipoles of the N-terminal ends near the central dehydrated anion-binding region and provide backbone amides to coordinate transient anions.

“Breaks in these helices position the positive dipoles of the N-terminal ends of the helices near the central dehydrated anion-binding region of the protein, and also provide backbone amides to coordinate the transient anions (Figure 3d).”

The cell can be rescued from fluoride by producing one or both of two non-homologous classes of transport proteins.

“Ultimately, the cell can be rescued from fluoride by producing one or both of two non‐homologous classes of transport proteins (Stockbridge et al., 2012; Ji et al., 2014; Last et al., 2018).”

One type of fluoride transport is passive, gradient-controlled passage via Fluc-F− channels.

“One such type of transport is a passive, gradient‐controlled passage of fluoride via what is known as Fluc‐F− channels.”

The other class are F−/H+ antiporters that act similarly to multi-drug resistance transporters and use a proton gradient or ATP hydrolysis to expel fluoride against a gradient.

“The other class are F−/H+ antiporters that act similarly to multi‐drug resistance transporters and use a proton gradient or ATP hydrolysis to expel fluoride against a gradient (Fig. 5).”