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

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 identified additional binding sites along the permeation pathway. We used this information to develop an oriented system for planar lipid bilayer electrophysiology and observed 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.

N-terminal Transmembrane-Helix Epitope Tag for X-ray Crystallography and Electron Microscopy of Small Membrane Proteins.

Structural studies of membrane proteins, especially small membrane proteins, are associated with well-known experimental challenges. Complexation with monoclonal antibody fragments is a common strategy to augment such proteins; however, generating antibody fragments that specifically bind a target protein is not trivial. Here we identify a helical epitope, from the membrane-proximal external region (MPER) of the gp41-transmembrane subunit of the HIV envelope protein, that is recognized by several well-characterized antibodies and that can be fused as a contiguous extension of the N-terminal transmembrane helix of a broad range of membrane proteins. To analyze whether this MPER-epitope tag might aid structural studies of small membrane proteins, we determined an X-ray crystal structure of a membrane protein target that does not crystallize without the aid of crystallization chaperones, the Fluc fluoride channel, fused to the MPER epitope and in complex with antibody. We also demonstrate the utility of this approach for single particle electron microscopy with Fluc and two additional small membrane proteins that represent different membrane protein folds, AdiC and GlpF. These studies show that the MPER epitope provides a structurally defined, rigid docking site for antibody fragments that is transferable among diverse membrane proteins and can be engineered without prior structural information. Antibodies that bind to the MPER epitope serve as effective crystallization chaperones and electron microscopy fiducial markers, enabling structural studies of challenging small membrane proteins.

Membrane Exporters of Fluoride Ion.

Microorganisms contend with numerous and unusual chemical threats and have evolved a catalog of resistance mechanisms in response. One particularly ancient, pernicious threat is posed by fluoride ion (F-), a common xenobiotic in natural environments that causes broad-spectrum harm to metabolic pathways. This review focuses on advances in the last ten years toward understanding the microbial response to cytoplasmic accumulation of F-, with a special emphasis on the structure and mechanisms of the proteins that microbes use to export fluoride: the CLCF family of F-/H+ antiporters and the Fluc/FEX family of F- channels.

Membrane Protein Production in Escherichia coli.

Escherichia coli is the workhorse of the structural biology lab. In addition to routine cloning and molecular biology, E. coli can be used as a factory for the production of recombinant membrane proteins. Purification of homogeneous samples of membrane protein expressed in E. coli is a significant bottleneck for researchers, and the protocol we present here for the overexpression and purification of membrane proteins in E. coli will provide a solid basis to develop lab- and protein-specific protocols for your membrane protein of interest. We additionally provide extensive notes on the purification process, as well as the theory surrounding principles of purification.

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

Fluc family fluoride channels are assembled as primitive antiparallel homodimers. Crystallographic studies revealed a cation bound at the center of the protein, where it is coordinated at the dimer interface by main chain carbonyl oxygen atoms from the midmembrane breaks in two corresponding transmembrane helices. Here, we show that this cation is a stably bound sodium ion, and although it is not a transported substrate, its presence is required for the channel to adopt an open, fluoride-conducting conformation. The interfacial site is selective for sodium over other cations, except for Li+, which competes with Na+ for binding, but does not support channel activity. The strictly structural role fulfilled by this sodium provides new context to understand the structures, mechanisms, and evolutionary origins of widespread Na+-coupled transporters.

Cork-in-Bottle Occlusion of Fluoride Ion Channels by Crystallization Chaperones.

Crystallization of dual-topology fluoride (Fluc) channels requires small, soluble crystallization chaperones known as monobodies, which act as primary crystal lattice contacts. Previous structures of Flucs have been solved in the presence of monobodies that inhibit fluoride currents in single-channel electrophysiological recordings. These structures have revealed two-fold symmetric, doubly bound arrangements, with one monobody on each side of the membrane. The combined electrophysiological and structural observations raise the possibility that chaperone binding allosterically closes the channel, altering the structure from its conducting form. To address this, we identify and solve the structure with a different monobody that only partially blocks fluoride currents. The structure of the channel-monobody complex is asymmetric, with monobody bound to one side of the channel only. The channel conformation is nearly identical on the bound and uncomplexed sides, and to all previously solved structures, providing direct structural evidence that monobody binding does not induce local structural changes.

Characterization of the Inward- and Outward-Facing Substrate Binding Sites of the Prokaryotic Aspartate Transporter, GltPh.

Crystal structures of the prokaryotic aspartate transporter, GltPh, have provided important insights into the mechanism of amino acid transport by GltPh and related eukaryotic members of the glutamate transporter family (SLC1A family). Identification of inhibitors of GltPh can provide valuable tools for understanding the molecular basis for substrate and inhibitor specificity and selectivity of SLC1A members, but at present, few inhibitors of GltPh have been identified. We have screened a collection of commercially available aspartate analogues and identified new transportable and nontransportable GltPh inhibitors. We have explored the inhibition profile of GltPh by utilizing a thiol modification assay that isolates sided populations of the transporters reconstituted in liposomes to determine if any aspartate analogues display a preference for either the inwardly or outwardly directed binding sites. Here, we have characterized several new inhibitors of GltPh and identified three ß-carbon-substituted molecules that display a strong preference for the outwardly directed binding site of GltPh.

Transport rates of a glutamate transporter homologue are influenced by the lipid bilayer.

The aspartate transporter from Pyrococcus horikoshii (GltPh) is a model for the structure of the SLC1 family of amino acid transporters. Crystal structures of GltPh provide insight into mechanisms of ion coupling and substrate transport; however, structures have been solved in the absence of a lipid bilayer so they provide limited information regarding interactions that occur between the protein and lipids of the membrane. Here, we investigated the effect of the lipid environment on aspartate transport by reconstituting GltPh into liposomes of defined lipid composition where the primary lipid is phosphatidylethanolamine (PE) or its methyl derivatives. We showed that the rate of aspartate transport and the transmembrane orientation of GltPh were influenced by the primary lipid in the liposomes. In PE liposomes, we observed the highest transport rate and showed that 85% of the transporters were orientated right-side out, whereas in trimethyl PE liposomes, 50% of transporters were right-side out, and we observed a 4-fold reduction in transport rate. Differences in orientation can only partially explain the lipid composition effect on transport rate. Crystal structures of GltPh revealed a tyrosine residue (Tyr-33) that we propose interacts with lipid headgroups during the transport cycle. Based on site-directed mutagenesis, we propose that a cation-π interaction between Tyr-33 and the lipid headgroups can influence conformational flexibility of the trimerization domain and thus the rate of transport. These results provide a specific example of how interactions between membrane lipids and membrane-bound proteins can influence function and highlight the importance of the role of the membrane in transporter function.