On April 8, the research group led by Yan Zhao at the Institute of Biophysics, Chinese Academy of Sciences, published a research paper in the international academic journal "Nature Structural & Molecular Biology". This study utilized single-particle cryo-electron microscopy to decode for the first time the transport regulation mechanism of the high-affinity choline transporter 1 (CHT1).
CHT1-mediated choline reuptake is the rate-limiting step in acetylcholine synthesis. Its aberrant expression and functional impairment can trigger a variety of diseases, such as hereditary motor neuron diseases, myasthenia syndromes, arteriosclerosis, depression, and Alzheimer’s disease. However, the key sites for CHT1's recognition of choline, the structural basis for its conformational changes, and the inhibition mechanism of its transport activity by the small molecule HC-3 are yet to be elucidated.
In the outward-facing structure of CHT1 bound with HC-3, HC-3 presents a rod-like shape and inserts from the outside into the substrate binding pocket, locking the protein in an outward-open state. In the inward-facing choline-bound state, the intracellular pocket of CHT1 opens inward, with choline still stabilized by a tryptophan triad. In the inward-facing substrate-unbound state, the tryptophan triad pocket undergoes a conformational change, exposing choline to the intracellular solvent side and thereby leading to the release of choline from the substrate binding pocket.
Moreover, the short intracellular helix IH1 also plays a crucial role in the CHT1 transport process. In the outward-facing CHT1 structure, the IH1 helix participates in maintaining conformational stability. When the substrate binds to CHT1, converting it from an outward to an inward state, the IH1 helix is released, exposing the intracellular pocket of CHT1 and facilitating transport. Further experiments indicate that the IH1 helix deletion mutant completely loses substrate transport activity and is unable to transition from an inward to an outward state.
In summary, this study has decoded the high-resolution structures of CHT1 in the outward HC-3-bound state, the inward substrate-unbound state, and the inward choline-bound state. Through a series of experimental methods such as radioactive isotope tracing, substrate uptake experiments, and molecular dynamics simulations, it elucidated the molecular model of CHT1 recognition and transport of choline and the mechanism of HC-3 inhibiting transport activity. This research provides a structural basis for the development of novel CHT1-targeted drugs.
