Biochemical and Functional Characterization of a Novel Prokaryote Ligand-Gated Ion Channel CLIC

Abstract

Crystal structures derived from acetylcholine binding proteins, prokaryotic and invertebrate pentameric ligand-gated ion channels (pLGIC) have proven to be valuable tools to study underlying mechanisms concerning ligand binding, channel gating and ion permeation. The aim of this study is to further expand this knowledge by identifying suitable targets for structural studies. Here we report the identification of a novel prokaryotic pLGIC, referred to as CLIC. CLIC is readily expressed in E.coli cells and can be extracted from the cell membrane using detergents in a homogenous and monodisperse state. Purification using a maltose-binding protein affinity tag and size-exclusion chromatography yields milligram quantities of the receptor in a biochemically stable state. CLIC produces crystals within one week but the crystal diffraction quality is limited due to anisotropic diffraction, which prevents structural elucidation. Extensive optimization of crystal growth in the presence of different lipids and detergents has failed to improve crystal diffraction. We will describe the biochemical properties of nanobody- and Fab-based complexes of CLIC to enhance crystal quality. In parallel, we have conducted a ligand screening using CLIC expressed in Xenopus oocytes. We discovered several ligands that activate CLIC and can be used as future tools in crystallization trials. Together, these results open perspectives for the structure determination of novel prokaryote pLGICs.