Formate translocation by the FocA channel: How the N-terminal domain modulates anion specificity

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Fermenting enterobacteria growing at pH > 6.5 translocate formate from the cytoplasm, where it is generated by pyruvate formate-lyase (PflB), to the periplasm. At pH < 6.5, formate is taken back up into the cell, where it is disproportionated to CO2 and H2 by the formate hydrogenlyase complex. Reversible translocation of formate across the cytoplasmic membrane is carried out by FocA, which belongs to the evolutionarily ancient formate-nitrite transporter (FNT) family, members of which are 150 kDa pentameric channels that show structural similarity to tetrameric aquaporins. Each protomer of FocA has a hydrophobic pore that in vivo reversibly translocates formate; how this is achieved, and how ‘gating’ of the channel occurs, are not completely understood. Investigation of this translocation process is the focus of this proposal. The hydrophobic pore of FocA contains a centrally localized histidine residue (H209), which is the only charged amino acid in the pore. This histidine residue is conserved in roughly 99% of all FNT proteins. The only known exceptions have either asparagine or glutamine at this position. Our recent findings indicate that when H209 is converted to either of these non-protonable amide residues, FocA becomes an exclusive formate efflux channel. This indicates H209 is necessary for formate uptake by FocA and suggests a physiological rationale for pH-dependent formate translocation in E. coli. Amino acid T91 is also highly conserved within the hydrophobic pore and it is located in proximity to H209. T91 is also essential to allow reversible anion permeation through the pore. We have recently determined the 3.1 Å structure of the complete FocA channel (collaboration with P. Kastritis in Halle) using cryo-EM and this supports a role for H209 and T91 in controlling anion passage. As nearly all FNT channels have a number of conserved residues within the hydrophobic pore of each protomer, this suggests anion specificity is achieved by another mechanism. We have shown recently that the cytoplasmically localized N-terminal domain of FocA is essential for its in vivo function. Moreover, our data indicate that PflB specifically binds to this N-terminal domain, suggesting this interaction might control anion access to the translocation pore. The N-terminal domains of different FNTs show no conservation in sequence or secondary structure. This suggests that control of FNT pore gating by the N-terminal domain might represent a common mechanism spanning the complete superfamily. We plan domain-swapping experiments with three different FNTs to test this hypothesis. Thus, the overall aims of this proposal are to elucidate the functional roles of conserved pore residues in formate translocation and to determine how the N-terminal domain acts to control reversible formate passage through the FocA pore.