Molecular Interactions between a Fluoride Ion Channel and Synthetic Protein Blockers

Abstract

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.

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

“…The variable loop's polar residues appear to be solvated by bulk water in the vestibule. This dearth of putative H-bonding interactions between S8 and Bpe is a marked departure from previously observed Bpe/monobody interfaces, which typically involve six or seven H-bonded or salt bridge pairs, many within the vestibule, that contribute substantially to the binding affinity ( Turman and Stockbridge, 2017 , Turman et al., 2018 ).…”

“…The chaperones required to obtain Fluc crystals (engineered "monobodies" derived from a human fibronectin domain and selected from diversified phage-display libraries; Koide et al., 2009 , Koide et al., 2012 ) bind to both ends of the channel protein as in the crystal structures of Figure 1 . All previously published structures of Fluc channels, representing four different channel/monobody complexes, show two monobodies bound per channel, one on each end, apparently occluding the wide aqueous vestibules through which ions enter and leave the F – -selective transport pathways ( Last et al., 2016 , Last et al., 2017 , Stockbridge et al., 2015 , Turman et al., 2018 ). This doubly bound state is consistent with electrophysiological recordings, which show that, upon binding from either side, these monobodies render the channels nonconductive to F – ions, an inhibitory process commonly called "block" ( Stockbridge et al., 2014 , Turman et al., 2015 , Turman and Stockbridge, 2017 ).…”

An Engineered E. coli Strain for Direct in Vivo Fluorination

“…Lysates and supernatants from two replicates were assayed by HPLC and also 19 F-NMR, which directly reports fluorinated products. The lysates in both of fluorinase containing strains MK015 and MK019 showed clear 19 F-NMR signals associated with 5'-FDA as the only organo-fluorine product whereas the corresponding negative controls (MK016 and MK020) without the flA1 gene, did not.…”

“…A series of experiments (see SI for details) explored incubations of engineered E. coli's with and without samT expression and crcB gene active or deleted. Cell extracts were evaluated by 19 F-NMR ( Figure 2). No 5'-FDA was detected in MK004 and MK010, both of which express flA1 but not samT, with MK010 having an inactivated fluoride efflux channel.…”

“…[18] Several studies have revealed a wide variety of proteins that play a role in managing high fluoride ion concentrations in bacterial or eukaryotic hosts. [15,[19][20][21] These are transmembrane channels that serve as pumps expelling fluoride which has entered passively as HF. E. coli employs such a channel protein termed CrcB which it uses to achieve fluoride resistance.…”