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

Abstract

Summary 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 electrohysiological 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.

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

“…We propose either that the densities observed in the crystal structure represent partially occupied fluoride sites or that the monobodies used as crystallization chaperones alter the electrostatic landscape in the pore, increasing ion occupancy. Indeed, in crystal structures of Fluc-Bpe with a monobody occupying only one side of the channel, each pore contained only one fluoride density, rather than two, in the polar track ( McIlwain et al, 2018 ).…”

“…Crystal structures of representative Fluc channels from Bordetella pertussis (Fluc-Bpe) and an Escherichia coli virulence plasmid (Fluc-Ec2) provide an opportunity to understand the molecular basis for anion permeation in the Flucs ( Stockbridge et al, 2015 ; McIlwain et al, 2018 ). The protein possesses two deep, aqueous vestibules with an electropositive character due to an absolutely conserved arginine sidechain and a deeply buried sodium ion at the center of the protein ( Turman and Stockbridge, 2017 ; McIlwain et al, 2020 ).…”

Development and Differentiation in Monobodies Based on the Fibronectin Type 3 Domain

“…The small size (~10 kDa molecular weight) of individual FN3 domains confers increased tissue distribution and reduced half-life, whilst the absence of disulphide bonds improves stability in cytosolic environments and facilitates the introduction of non-native cysteines. The applications of monobodies in fundamental science discovery have been covered elsewhere [3], including for blocking of multiple sites on a target protein [54], altering activity through allosteric modulation [55], and as crystal chaperones [56]. However, more recently the monobody domain has found unique applications in the fields of therapeutics and diagnostics.…”

Development and Differentiation in Monobodies Based on the Fibronectin Type 3 Domain

“…The small size (~10kDa molecular weight) of individual FN3 domains confers increased tissue distribution and reduced half-life, whilst the absence of disulphide bonds improves stability in cytosolic environments and facilitates the introduction of non-native cysteines. The applications of monobodies in fundamental science discovery have been covered elsewhere [3], including for blocking of multiple sites on a target protein [53], altering activity through allosteric modulation [54], and as crystal chaperones [55]. However, more recently the monobody domain has found unique applications in the fields of therapeutics and diagnostics.…”