Fluoride/proton antiporters of the CLCF family combat F– toxicity in bacteria by exporting this halide from the cytoplasm. These transporters belong to the widespread CLC superfamily but display transport properties different from those of the well-studied Cl–/H+ antiporters. Here, we report a structural and functional investigation of these F–-transport proteins. Crystal structures of a CLCF homolog from Enterococcus casseliflavus are captured in two conformations with simultaneous accessibility of F– and H+ ions via separate pathways on opposite sides of the membrane. Manipulation of a key glutamate residue critical for H+ and F– transport reverses the anion selectivity of transport; replacement of the glutamate with glutamine or alanine completely inhibits F– and H+ transport while allowing for rapid uncoupled flux of Cl–. The structural and functional results lead to a ‘windmill’ model of CLC antiport wherein F– and H+ simultaneously move through separate ion-specific pathways that switch sidedness during the transport cycle.
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We thank members of the laboratory of C.M. for advice and criticism, and we acknowledge with ambiguous gratitude frequent and interminable discussions with A. Accardi. We are unambiguously grateful to the beamline scientists for their expert help with resources at the Advanced Light Source, a DOE User Facility, contract DE-AC02-05CH11231. This project was supported in part by NIH grants R01-GM107023 (C.M.), U54-GM087519 (S.K.), and S10OD021832 (for ALS beamline 5.0.1).
The authors declare no competing interests.
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a, c. F- ion omit mFo-DFc maps (green mesh) in WT Eca contoured to 4 σ (a) or E118Q contoured at 3.5 σ (c). Glug sidechain (orange stick) is also indicated in all panels b, d. Stereo images of 2mFo-DFc density contoured to 1.2 σ (grey) in WT (b) or E118Q (d).
a. Overlay of WT Eca subunit (cyan) with Cl-/H+ antiporter CLC-ec1 (grey, PDB# 1OTS). b. Alignment of ion-coupling regions of WT Eca with CLC-ec1 E148Q (PDB# 1OTU), with mutated Glug sidechain in Up-position. Positions of Eca F- (magenta) and CLC-ec1 Cl- (yellow) are also indicated.
F- titrations (from 3 repeats each) for WT (left) and V319G (right). Solid lines represent KD = 0.20 mM, 1.0 mM, respectively.
a. F- ion difference density. mFo-DFc map refined in the absence of F- (green mesh) contoured to 4 σ. b. Stereo view of 2mFo-DFc density contoured to 1.2 σ (grey) in the region surrounding Glug (orange stick). c. Alignment of ion-coupling regions of V319G Eca with the CLC Cl-/H+ antiporter from C. merolae (brown ribbon, yellow Cl-, PDB 3ORG).
Proposed pathway of Glug from Up to Down conformations in Step 1 of transport cycle of Fig. 6. Rotamers shown allow F-ex to remain bound during the transition. This motion would likely require movement of F154, shown at right. Y396 is also shown below.
Complete sequence of the monobody “X1” used for crystallization. Residues in red are removed upon TEV cleavage, leaving the black residues as the protein used to complex Eca. Residue numbering in the structures is based on the cleaved protein. Penultimate residue (underlined) is an arginine in the V319G structure.
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Last, N.B., Stockbridge, R.B., Wilson, A.E. et al. A CLC-type F-/H+ antiporter in ion-swapped conformations. Nat Struct Mol Biol 25, 601–606 (2018). https://doi.org/10.1038/s41594-018-0082-0
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