Molecular Interactions between a Fluoride Ion Channel and Synthetic Protein Blockers.

Fluoride ion channels of the Fluc family selectively export F- ions to rescue unicellular organisms from acute F- toxicity. Crystal structures of bacterial Fluc channels in complex with synthetic monobodies, fibronectin-derived soluble ß-sandwich fold proteins, show 2-fold symmetric homodimers with an antiparallel transmembrane topology. Monobodies also block Fluc F- current via a pore blocking mechanism. However, little is known about the energetic contributions of individual monobody residues to the affinity of the monobody-channel complex or whether the structural paratope corresponds to functional reality. This study seeks to structurally identify and compare residues interacting with Fluc between two highly similar monobodies and subjects them to mutagenesis and functional measurements of equilibrium affinities via a fluorescence anisotropy binding assay to determine their energetic contributions. The results indicate that the functional and structural paratopes strongly agree and that many Tyr residues at the interface, while playing a key role in affinity, can be substituted with Phe and Trp without large disruptions.

Two-sided block of a dual-topology F- channel.

The Fluc family is a set of small membrane proteins forming F(-)-specific electrodiffusive ion channels that rescue microorganisms from F(-) toxicity during exposure to weakly acidic environments. The functional channel is built as a dual-topology homodimer with twofold symmetry parallel to the membrane plane. Fluc channels are blocked by nanomolar-affinity fibronectin-domain monobodies originally selected from phage-display libraries. The unusual symmetrical antiparallel dimeric architecture of Flucs demands that the two chemically equivalent monobody-binding epitopes reside on opposite ends of the channel, a double-sided blocking situation that has never before presented itself in ion channel biophysics. However, it is not known if both sites can be simultaneously occupied, and if so, whether monobodies bind independently or cooperatively to their transmembrane epitopes. Here, we use direct monobody-binding assays and single-channel recordings of a Fluc channel homolog to reveal a novel trimolecular blocking behavior that reveals a doubly occupied blocked state. Kinetic analysis of single-channel recordings made with monobody on both sides of the membrane shows substantial negative cooperativity between the two blocking sites.