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 monobodybinding 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.ion channel | monobody | block | cooperativity S everal years ago, Baker et al.(1) discovered that many microorganisms harbor in their membranes anion-exporter proteins that keep cytoplasmic F − below the toxic concentrations encountered throughout the aqueous environment. The novelty of this previously unsuspected microbial physiology is mirrored in the unusual molecular architecture of one class of these exporters, the Fluc family. Flucs are highly F − -selective ion channels built as dual-topology homodimers, wherein the paired subunits of the functional channel assemble in antiparallel transmembrane topology (2, 3). This architecture demands that, if the twin subunits adopt identical conformations, the channel must present structurally identical ion entryways to the two sides of the membrane, in sharp contrast to the parallel assembly of conventional multisubunit membrane proteins. Antiparallel assembly of Flucs was established definitively (2) by the use of "monobodies," small fibronectindomain proteins of known structure engineered by random variation of amino acid sequences and selected in combinatorial libraries as nanomolar affinity-specific binders (4). In double-sided perfusion experiments, single Fluc channels were shown to be blocked with similar kinetics by monobodies added separately to the internal or external aqueous solution. The channel thus presents to each side of the membrane identical epitopes for the blocker, as required of symmetrical, antiparallel assembly (Fig. 1A), a circumstance that naturally raises the question can monobodies occupy both blocking sites simultaneously?We address this question by direct binding and single-channel experiments with monobody on both sides of the channel. The results reveal a previously unobserved "trimolecular" channelblocking beha...
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 β-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.
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