Easy passage for fluoride ions revealed in new structure
A collaboration between Simon Newstead and researchers in the States has led to the discovery of the structure of an ion channel that rids cells of toxic fluoride ions.
Fluc channel from Bordetella pertussis (Bpe). Homodimer viewed parallel to the membrane, with colouring according to the transmembrane helix schematic on left (Click to enlarge)
Simon Newstead, working with Christopher Miller and Randy Stockbridge at Brandeis University and Shohei Koide at the University of Chicago, have published the work on the dual-topology ion channel Fluc in Nature (1).
The Fluc family of ion channels is found in organisms from bacteria to eukaryotes. They export fluoride ions, which are toxic because they inhibit certain phosphoryl-transfer enzymes, out of the cell. Microorganisms in particular are continuously exposed to fluoride ions, which are present in soil and water.
Recent work from Professor Miller's lab on the Fluc channels showed that they have unusual properties (2, 3). They have a dual-topology architecture - two identical subunits are arranged in an inverted dimer orientiation to form the active channel, an assembly not previously seen in ion channels but common in multidrug transporters. The channels also show extreme selectivity towards fluoride ions over the more abundant chloride ions.
Whilst Professor Miller was in Oxford as a Newton Abraham Visiting Professor in 2013-14, his and Professor Newstead's paths crossed. This led to collaboration on the X-ray structure of the Fluc channel.
Bpe with S7 monobodies bound. Variable sequences of the monobodies are in shown cyan with ribbon or mesh representation (Click to enlarge)
Professor Newstead's own work facilitated the discovery of the crystal structure, as he explains: 'Crucial to the success of the project was the new lipidic cubic phase crystallisation method for crystallising membrane proteins. This is a facility we have set up in the department thanks to a grant from the EPA Cephalosporin Fund.'
The project required another collaboration, with Professor Shohei Koide. His lab works on synthetic protein science and specialises in making monobodies - artificial binders engineered to target proteins of interest. The monobodies bind to the protein and help to form crystallisation contacts.
With these two approaches in hand, the researchers successfully crystallised the Fluc fluoride channel.
The structure reveals that the dual-topology ion channel has two pores for fluoride ion diffusion, each pore constructed by side chains from both monomers. The double-barreled structure is unique amongst ion channels. The researchers propose that a Na+ ion, located in the middle of the membrane, stabilises the interface between the subunits - presenting an entirely new folding mechanism for ion channels.
Arrangement of fluoride ion and side chains within Bpe, with Phe82, Ser112 (fluoride ion 1), Asp43, Ser108, and Phe85 (fluoride ion 2) shown as sticks (Click to enlarge)
The structure also highlights the unique selectivity mechanism adopted by the channel. Diffusion of the fluoride ion through the pores requires an interaction between the ion and a conserved asparagine side chain, an interaction that is specific to fluoride. A 'phenylalanine box', an arrangement of two pairs of phenylalanine rings, is thought to play an important role. It helps to attract fluoride ions and stablises their dehydrated state. Once there, they interact with the asparagine residue.
The researchers suggest that a rotameric switch of side chains, rather like a turnstile, is required to allow fluoride ions to diffuse down down their electrodiffusive potential across the membrane.
Professor Newstead comments about the unusual mechanism: 'Ion channels are well known for using a narrow pore to confer selectively, but in this case, there is no pore as such - rather, the interaction with asparagine side chains is key. This is similar to the mechanism used by transporters, consistent with the similar architecture.'