Structural insights into a conserved mechanism of choline translocation through CHT

Abstract

The essential nutrient choline is critical for cellular homeostasis across all domains of life. In humans, choline uptake in cholinergic neurons for its recycling into acetylcholine is mediated by the high-affinity Na-dependent transporter SLC5A7 (also known as CHT1). Prokaryotes also depend on choline as an osmo-protectant and as metabolite, raising the possibility that bacteria also possess choline transporters akin to CHT1. Here, we present a bacterial Na+-dependent choline transporter (sfCHT) with high sequence identity to CHT1. sfCHT transport activity can be blocked by the choline transport inhibitor hemicholinium-3. Cryo-EM structures of Na+- and choline-bound sfCHT reveal a 14 transmembrane helix topology with a LeuT-fold architecture and Na+ coordination geometry similar to CHT1. Captured in an inward-facing conformation, in sfCHT choline is found at a site near the cytoplasmic side. Computational analysis and transport assays reveal local conformational changes along a choline translocation pathway to the cytosolic site. Transport assays with CHT1 variants, carrying substitutions at conserved residues along the proposed translocation pathway in sfCHT, reveal a conserved mechanism of choline transport between the bacterial and human choline transporters.