Structure and mechanism of the FocA formate channel: Discovery of an organic ion channel

Abstract

Transport of charged and polar solutes across membranes is fundamental to cellular organisms, and studies on these processes are elemental to the understanding of a wide variety of biological fields ranging from cellular metabolism to neurology. Integral membrane channel proteins represent basic systems of solute transport, catalyzing the translocation of their substrates down a concentration gradient without the need for external energy sources. Nevertheless, channel proteins exhibit a high degree of substrate selectivity and yet can facilitate ionic flux rates that often approach the limits of diffusion.

In anaerobic environments, microorganisms convert up to one third of metabolized glucose into the simple carboxylate ion, formate. In the Escherichia coli bacterium, this metabolite is transported across the inner membrane through the FocA protein, which serves as the founding member in the formate/nitrite transporter (FNT) family encompassing over 1500 homologues in bacteria, yeast and simple eukaryotes. Additionally, a nitrite transporting variant of the FNT family has been shown in mice to play a role in Salmonella pathogenesis and may provide a paradigm for bacterial defense against reactive nitrogen species released by the host.

High resolution structural data for any FNT family members has been unavailable, and the mechanism of substrate translocation has not been determined. In this work, the successful purification, crystallization and subsequent structure elucidation of the novel FocA protein from Vibrio cholerae structure to 2.1 Å resolution is described. A supplemental crystal structure shows the location of two substrate molecules near a central and conserved region of the cytoplasmic pore, and biochemical assays support activity of the protein.

The tertiary protein structure exhibits a homopentameric organization, however the monomeric topology and fold is represented in the tetrameric aquaporin family of water and glycerol channels. Within the crystallographic pentamer, a conserved half-membrane spanning pore helix adopts different conformations. In addition, the location of substrate molecules in this region indicates that the FocA selectivity filter is formed on the cytoplasmic side of the protein and that lateral gating of the pore region is activated by the presence of formate molecules. A hypothesis for selectivity and gating in the FNT family is presented.